Lanthanide complexes for crystallising biological macromolecules and determining the crystallographic structure thereof

ABSTRACT

The invention relates to cationic complexes made up of a lanthanide ion Ln3+ and a ligand of formula (I): 
                         
with X, Y and R1 as defined in claim  1 , and to the salts thereof with an anion, the solvates and hydrates thereof;
 
with the exception of cationic complexes made up of a lanthanide ion Ln3+ and a ligand of one of formulae (I.1) or (I.4) as defined in claim  1 , and the salts, solvates and hydrates thereof. The invention also relates to the use of such a complex or of a cationic complex made up of a lanthanide ion Ln3+ and a ligand of formula (I.1) or (I.4) as defined in claim  1 , or of one of the salts thereof with an anion, the solvates or hydrates thereof, as an aid to the crystallisation of a biological macromolecule, as well as to crystallisation methods and methods for analysing or determining the structure of a biological macromolecule.

The present invention relates to the technical fields of crystallization and crystallography. More specifically, the invention concerns new lanthanide complexes which can be used as phasing agents for the determination of the crystal structure of biological macromolecules, but also as an aid for their crystallization. The invention is also intended to be used in these fields and the crystallization and determination of structural data which implement them.

The resounding success of the full determination of the human genome in 2000 paved the way for an even broader area of research: structural genomics, which involves determining the structure of proteins to understand the relationships between their structure and function. Due to the number and variety of proteins available, this is a huge project whose scope in terms of scientific and medical benefits is invaluable. Today, the two tools for this structural resolution are crystallography and nuclear magnetic resonance (NMR). These are two complementary techniques with advantages and limitations: (i) NMR allows protein solution analysis but requires isotopic enrichment; (ii) crystallography allows faster determination of structures but remains dependent on the preparation of good quality crystals. Currently, the research effort is focused on the development of very large instruments to the detriment of the search for new methodologies.

In this area, several successive difficulties have to be overcome. The first difficulty is in the field of biology and concerns the preparation and purification of proteins of interest and only 18% of cloned proteins pass this stage. The second difficulty concerns the crystallization stage, since again only 20% of the purified proteins can be crystallized and only half of these crystals will allow the structure to be determined by X-ray diffraction. On balance, only 1.8% of cloned proteins are structurally characterized and this, at the cost of considerable investment in terms of time and human and financial resources.

A crystal of a protein, or more generally a biological macromolecule, consists of a regular and periodic stacking of the molecules that make up the protein. Stacking is maintained by contacts within the crystal (hydrogen bonds, salt bridges, hydrophobic contacts). Obtaining a crystal from a biological macromolecule is a key step in determining its structure by X-ray crystallography.

It should be noted that the crystallization processes of biological macromolecules are still far from being understood and that current approaches are based on an empirical trial and error approach.

One of the important notions to consider in crystallogenesis is the concept of solubility. In solution, a macromolecule is surrounded by a solvent-based layer that has a repellent and insulating effect and avoids self aggregation and precipitation. Crystallization goes against this effect, since it involves moving away from the solubility conditions of the biological macromolecule by adding a precipitating agent, also called a crystallizing agent. For the purposes of this description, “precipitating agent” and “crystallizing agent” are used interchangeably. crystallizsing/precipitant means a molecule or mixture of molecules which, under certain conditions, help to form a crystal of biological macromolecules.

The general principle of crystallization is based on the diagram presented in FIG. 1, which corresponds to the diagram produced by Mirjam Leunissen and appearing on http://people.ds.cam.ac.uk/ml527/publications/assets/leunissen-literature-research.pdf.

The two main steps that occur in the crystallization process are: nucleation and crystal growth. The crystallizing agent induces a change in solubility and therefore in the position on the diagram as a function of time. There are different crystallization methods using a crystallizing agent. The crystallizing agent induces a change in solubility and therefore in the position on the diagram as a function of time. There are different crystallization methods using a crystallizing agent.

The aim during crystallization is to play on the concentrations of biological macromolecule and crystallizing agent in order to reach the nucleation zone represented on the diagram in FIG. 1. When this zone is reached, nuclei (nucleation points) can form. These nuclei are more or less organized aggregates of about a hundred macromolecules. The formation of these “crystalline” departures reduces the concentration of soluble macromolecule. This is the beginning of crystal growth. Other macromolecules are added to this nuclei and form microcrystals that will grow over time. There are three major classes of crystallizing agents:

-   -   salts,     -   organic solvents (ethanol, dioxane, MPD for methylpentanediol         etc.)     -   polymers (PEG (Poly-Ethylene Glycol) with molecular weighting up         to 20000 g/mol, Jeffamine T etc.)

These agents have the effect:

-   -   to compete with the biological macromolecule for water, or more         generally the solvent in which the macromolecule is initially in         solution, and thus decrease the solubility of the macromolecule,         inducing its crystallization,     -   to shield charges on the surface of the macromolecule allowing         several macromolecules to get closer together,     -   to change the dielectric constant of the solvent and therefore         the forces between macromolecules     -   to favor phenomena of excluded volume type (hydrophobic         exclusion).         To obtain the crystallization of a protein, or more generally of         a biological macromolecule, an automated approach allowing a         screening of different commercially available crystallization         conditions (for example, Hampton Research crystallization kits).         Crystallization experiments are prepared by robots and the         crystallization phase is regularly monitored by microscope         robots that scan the appearance of the crystals. This         high-throughput approach has shifted the search for alternative         methodologies to promote crystallization, including finding         agents that promote crystallization without denaturing the         protein. Some works exist on this subject in the literature,         such as the use of natural compounds: mineral solids (Falini G.,         Fermani S., Conforti G., Ripamonti A. (2002); Acta Crystallogr D         Biol Crystallogr. 58, 1649-1652, McPherson, A. & Shlichta, P.         (1988), Science, 239, 385-387), substrate of biological origin         such as hair, horsehair, rat whiskers or dried algae (D'Arcy A.,         Mac Sweeney A. & Haber, A. (2003), Acta Crystallogr D Biol         Crystallogr. 59, 1343-1346), but also synthetic products         (Bijelic A., Molitor C., Mauracher S., G. Al-Oweini, R.,         Kortz U. & Rompel, A. (2014), Chem Bio Chem. 16, 233-241,         Pechkova, E. & Nicolini, C. (2002), J. Cell. Biochem. 85,         243-251, Sugahara, M. Asada, Y. Morikawa, Y. Kageyama, Y. &         Kunishima, N. (2008), Acta Crystallogr D Biol Crystallogr. 64,         686-695, Matar-Merheb, R. Rhimi, M. Leydier, A. Huché, F.         Galián, C. Desuzinges-Mandon, E. Ficheux, D. Flot, D.         Aghajari, N. Kahn, R. et al. (2011), PLoS ONE. 6, e18036).

In all these cases, crystalline growth occurs in contact with these “impurities”. The most successful work in this area is that of Naomi E. Chayen's team, which proposes the use of mesoporous materials, capable of inducing nucleation in pores after a concentration/aggregation stage within the pores (WO 02/088435). The same group also describes the use of MIP “molecular imprinted polymers” based on the same principle. These are polymers previously printed by the protein to be crystallized and which therefore contain traces of it. Added to the crystallization drop, a form interaction can occur that favors nucleation (Saridakis, PNAS, 2001, 108,11081). All these crystallization aid agents are solids forming a heterogeneous phase in the crystallization medium, each of which must therefore be added manually to the crystallization drop, which is incompatible with the use of robotic screening platforms, except for MIP.

Moreover, due to the gigantic size of biological macromolecules, such as proteins, the determination of their structure by X-ray diffraction involves solving the problem of crystallographic phases. This implies having a native crystal (pure protein) and/or a derivative crystal containing a heavy atom easily identifiable and allowing to remove the problem of phases, we speak of phasing agent. The most classical method, based on muitiwavelength anomalous diffraction (MAD) is based on the replacement of the sulphur of the amino acids methionines of the protein by a selenium atom. Used since the 1990s, this method has revolutionized macromolecular crystallography, but it involves the synthesis, purification, crystallization and resolution of the structure of analogous selenium proteins. This is a very time-consuming method. Other exogenous phasing agents can be used and are marketed, such as bromine, heavy metals (Pt, Au, Hg). In this context, R. Kahn showed in the early 2000s that lanthanides, which have some of the most intense anomalous effects, can be included as complexes in protein crystals, which allows the structure of the macromolecule to be resolved quickly if lanthanide is fixed (E. Girard, M. Stelter, P. L. Anelli, J. Vicat, R. Kahn, Acta. Cristallogr. D, 2003, D59, 118; E. Girard, P. L. Anelli, J. Vicat, R. Kahn, Acta. Cristallogr. D, 2003, 059, 1877). To date, five lanthanide complexes are marketed as phasant agents by NatX-ray.

It exists in the literature some phasing luminescent agents, such as functional derivatives of DPA₃ (FR 2 991 322 et ACIE 2008) or complexes of lanthanide directly grafted to the structure of proteins (X. C. Su, H. B. Liang, K. V. Loscha and G. Otting, J. Am. Chem. Soc., 2009, 131, 10352-10353).

Of all these derivatives, the tri-anionic complexes [(DPA)₃Ln]³⁻ seemed very promising because specific interactions with cationic amino acids and, in particular, arginine, were highlighted by some of the inventors of this patent application (E. Dumont, G. Pompidor, A. D'Aléo, J. Vicat, L. Toupet, R. Kahn, E. Girard, O. Maury, N. Giraud, Phys Chem Chem Phys 2013, 15, 18235-18242), and confirmed by NMR measurements (X. C. Su, H. B. Liang, K. V. Loscha, G. Otting, J. Am. Chem. Soc., 2009, 131, 10352-10353). Unfortunately, this compound has been shown to be unstable in a large number of commercial crystallization kits; (i) the presence of transition metals (Zn (II), Cu (II), Fe (II)) induces the destructuring of the complex, (ii) the presence of divalent alkaline earth salts (Ba (II), Ca (II), Mg (II)) induces immediate self-crystallization of the complex giving rise to false positives detection (crystals of complexes and not of crystals derived from proteins) (Doctoral thesis of R. Talon supported defended in Grenoble on Jun. 6, 2012).

Other solutions have proposed to introduce lanthanides into protein crystals to obtain a phasant effect. Nagem et al. (Nagem, R. A., Dauter, Z. & Polikarpov I, Acta Crystallogr. D, 2001, D57, 996-1002) proposed the use of lanthanide salts and the rapid soaking method (Dauter Z., Dauter, M. & Rajashankar, K. R. Acta Crystallogr. D, 2000, D56, 232-237). This method, originally developed for the haiogenide salts (NaCl, NaI, NaBr) consists of soaking the crystals in a highly concentrated salt solution (>1 mol L⁻¹) for a time of less than one minute. In the case of lanthanide salt, this method led to rapid crystal degradation. Purdy et al (Purdy M. D., Ge P., Chen 1, Selvin P. R., & Wiener, M. C. Acta Crystallogr. D, 2002, D58,1111-1117) proposed to use a covalent bond linking lanthanide complexes (where the ligand provides complete coordination of lanthanide) to the protein. The binding consists of a disulfide bridge formed between free cysteines of the protein and a thiol reactive function carried by the complex. Finally, Silvaggi et al. (Silvaggi, N. R., Martin, L. 1, Schwalbe, H., Imperiali, B., Allen, K. N. 1, Am. Chem. Soc, 2007 129,7114-7120) proposed the use of a “tag” fixing one or two lanthanides (LBT for Lanthanide-Binding Tag). LBT is based on a peptide sequence derived from calcium loops that can be introduced into proteins by conventional molecular biology techniques (Allen, K. N. Imperiali B., Current Opinion in Chemical Biology, 2010, 14, 247-254).

There is therefore a need for new phasing agents that do not require modification of the structure of biological macromolecules, the structure of which is to be determined. In addition, the invention proposes to provide phasing agents which are sufficiently stable in most conditions of crystallization of biological macromolecules and which also make it possible to broaden increase the possibilities of obtaining crystals allowing to obtain structural informations on the biological macromolecules of interest. In this context, the invention concerns cationic complexes consisting of a lanthanide Ln³⁺ and a ligand of formula (I):

-   -   in which: X and Y, which may be identical or different, each         independently represent —CH₂—(CH₂)_(n)—,         —CH₂—(CH₂)_(n)-A-CH₂CH₂— or —CH₂—CR₃R′₄—CH₂—, X being based on         the general formula (I) from left to right and Y being based on         the general (I) from right to left, with::         -   n which is equal to 1, 2 or 3;         -   A which is —NR₂—, —O(CH₂)₂O— or —O—, with R₂ which is a             hydrogen atom or a methyl group or —CH₂R₅, where R₅ is a             phenyl, pyridinyl or picolinyl;         -   R₃ represents a methyl group; and         -   R′₄ represents —NHR₄ with R₄ which represents —H, —CH₃,             —CH₂R₆; R₆ represents a phenyl or pyridinyl group;     -   R₁ represents:

-   -    with:         -   R₇ which represents —COO, —CONH₂, —CONHR₉, —PR₉OO⁻, or a             selected group chosen among:

-   -    with R₉ which represents a hydrogen atom, a methyl, ethyl or         phenyl group; and         -   R₈ which represents a hydrogen, fluorine, chlorine, bromine             or iodine atom, an —OH or —NH2 group;             with the proviso that at least one of the sequences X and Y             is different from —CH₂—(CH₂)_(n)—,     -   and their salts with an anion, their solvates and hydrates; with         the exception of cationic complexes consisting of a lanthanide         ion Ln³⁺ and a ligand having one of the following formulae (I.1)         to (I.5) and salts thereof with anion, solvates and hydrates         thereof:

The ligands of formula (I) comprise at least 7 coordination sites for a lanthanide ion Ln³⁺, or more, depending on the structures of X and Y. These coordination sites are located on the two nitrogen atoms represented on formula (I), on the two pyridines of the R₁, on the two R₇ groups, at least one coordination site is located on X and/or Y.

The complexes according to the invention, as well as the lanthanide complexes formed with ligands of the formulae (I.1) to (I.5), are cationic and have a positive charge greater than or equal to 1. They comprise a macrocyclic ligand incorporating several aromatic groups forming an open coordination sphere for the lanthanide ion. These aromatic groups, when the Ln³⁺ ion is Eu³⁺ or Tb³⁺, also act as antennas to sensitize the luminescence of lanthanide in the visible.

Furthermore, the complexes according to the invention, have the advantage of being water soluble and stable in most commercial crystallization media. In the context of the invention, these complexes have been shown to be of interest not only as a phasing agent when obtaining structural data, but also as an aid to crystallization. In particular, said complex is used as a nucleating agent and/or as a crystallizing agent in the crystallization of a biological macromolecule. Such applications had never been described or considered for lanthanide complexes formed with ligands of the formulae (I.1) to (I.5) already described in the literature (M. Mato-Iglesias et al., Inorg. Chem. 2008, 47, 7840-7851; Z. Palinkas et al., Inorg. Chem. 2009, 48, 8878-8889; A. Roca-Sabio et al., Dalton Trans, 2011, 40, 384-392 and M. Regueiro-Figueroa et al., Inorg. Chem. 2015, 54, 4940-4952 for ligand complexes (I.1) to (I.3) and A. Rodrigues-Rodrigues et al., Inorg. Chem. 2012, 51, 2509-2521, for ligand complexes (I.4) and (I.5)). Indeed, these complexes were prepared for fundamental studies of coordination chemistry of lanthanides (study of the stability of complexes formed with different lanthanides) and in the case of Gd, only applications as an MRI contrast agent were considered.

In an advantageous way, the complexes according to the invention, as well as their salts with anion, solvates and hydrates, are formed with a ligand corresponding to one of the following formulae:

with R₁, R₂, n and R₄ as defined for formula (I).

As an example of salts, complexes according to the invention or complexes formed with one of the ligands (I.1) to (I.5) which are applied in the context of the invention, one can cite salts of a cationic complex with an anion (or several anions, depending on the charge of the complex) chosen from: Cl⁻, Br⁻, I⁻, OH⁻, NO₃ ⁻, triflate, PF₆ ⁻, SbF₆ ⁻, B(Ph)₄ ⁻, BF₄ ⁻, sulphates, sulphonates, carbonates, phosphates, phosphonates and carboxylates. Sulphates and sulphonates may correspond to SO₄ ²⁻, HSO₃ ⁻ or R′SO₃ ⁻, carbonates to CO₃ ²⁻,HCO₂ ⁻ or R′CO₂ ⁻, phosphates and phosphonates to R′OPO₃ ²⁻ and R′PO₃ ²⁻ and carboxylates to R′CO₂ ⁻, with R′ which may be, in particular, an alkyl or aryl group, in particular an alkyl group containing 1 to 4 carbon atoms or a phenyl group. In the form of salts, the complexes, depending on the invention, may also be in the form of hydrate or solvates, i.e. with at least one water or solvent molecule in the lanthanide coordination sphere. Preferably, the complexes according to the invention, as well as their salts with anion, solvates and hydrates, are formed with a ligand corresponding to the formula (IA):

wherein n=1 and R₂=H, and R₁ is as defined for the compounds of formula (I).

In an advantageous way, in the ligands of formula (I), (IA) to (IF), R₁ represents:

with:

-   -   R₇ which represents —COO⁻ or —PR₉OO⁻ with R₉ qui representing         methyl or ethyl; or R₇ representing a group:

-   -   R₈ which represents a hydrogen, fluorine, chlorine, bromine or         iodine atom.

In the context of the invention, when a substituent represents a picolinyl group, it represents:

Preferably, the complexes according to the invention are chosen among the complexes of formula:

as well as their salts with anion, in particular their hydrochloride salt, solvates and hydrates. In an advantageous way, the complexes according to the invention or the complexes formed with one of the ligands (I.1) to (I.5) which find application in the context of the invention, as well as their salts with an anion, in particular their salt. hydrochloride, their solvates and hydrates, are formed with a lanthanide ion Ln³⁺, Ln being Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb or Lu, with Eu, Tb, Yb and Lu which are preferred.

The invention is also intended to use a complex depending on the invention, or a cationic complex consisting of a lanthanide ion Ln³⁺ and a ligand of the formulae (I.1) to (I.5) as previously defined, or a salt thereof with an anion, solvates or hydrates, as an aid to the crystallization of a biological macromolecule. In particular, said complex is used as a nucleating agent and/or as a crystallizing agent in the is crystallization of a biological macromolecule. All lanthanide complexes according to the invention or formed with a ligand of formula (I.1) to (I.5), are of interest for their nucleating or crystallizing effect. However, only terbium or europium complexes will exhibit luminescence properties in the visible. Also, if in the complex, Ln=Eu or Tb, the complex can also be used as a luminescent agent for the detection of crystals. The invention is also intended to use a complex according to the invention, or a cationic complex consisting of a lanthanide ion Ln³⁺ and a ligand of formula (I.1) to (I.5) as previously defined, or a salt thereof with an anion, solvates or hydrates, as an aid in obtaining structural data of a biological macromolecule. In particular, said complex is used as a phasing agent in the structural determination by X-ray diffraction. Again, if in the complex, Ln=Eu or Tb, the complex can also be used as an aid for positioning the crystal in an X-ray beam.

In particular, in the context of the invention, “biological macromolecule” means in particular peptides (sequence of less than 100 amino acids), proteins (sequence of at least 100 amino acids) and nucleic acids, in particular DNA or RNA. Such biological macromolecules shall in particular have an average molecular weight of more than 1 kDa.

The invention also has as its object the crystals of a biological macromolecule comprising a complex according to the invention or a cationic complex formed of a lanthanide ion Ln³⁺ and a ligand of formula (I.1) to (I.5) as previously defined, or a salt thereof with an anion, solvate or hydrate, called derivative crystals.

The invention also has as its object a process for crystallizing a biological macromolecule, preferably selected from peptides, proteins and nucleic acids, in particular DNA or RNA, comprising the following steps:

-   -   Having a solution comprising said biological macromolecule and a         complex according to the invention, or a cationic complex         consisting of a lanthanide ion Ln³⁺ and a ligand of formula         (I.1) to (I.5) as previously defined, or a salt thereof with an         anion, solvate or hydrate,     -   obtaining crystals of the macromolecule from said solution.

The solution used may also include a precipitating agent, other than the complex, chosen in particular from among those used in commercial crystallizsation kits, in particular from those listed in the tables presented in Annexes 2A or 2B, such as, for example, salts such as ammonium salts, sulphates (especially ammonium sulphate), acetates, phosphates, citrates (especially sodium citrate), formiates, tartrates such as sodium or potassium tartrate, double tartrate, sodium and potassium, chlorides, iodides and fluorides, for example NaCl; polymers such as polyethylene glycols and Jeffamine T; ethanol, dioxane, methylpentanediol, glycerol, isopropanol, 2-methyl-2,4-pentanediol.

In the solution, the complex is advantageously present at a concentration of 1 to 100 mM, preferably at a concentration of 1 to 25 mM, and even more favorably at a concentration of 10 mM±10%.

In particular, crystals are obtained by vapor diffusion crystallization, by dialysis, in batch or in a process of crystallization in the cubic phase of lipids.

The crystallization process, depending on the invention, can be integrated into a screening process or an optimization of the crystallization conditions of a biological macromolecule. The crystallization process according to the invention can be automated.

The invention shall also have as its subject matter a method of analyzing or determining the structure of a biological macromolecule comprising the following steps:

-   -   (a) having at its disposal a derivative crystal from the         biological macromolecule as defined in the invention,     -   (b) analyzing the crystalline structure of the biological         macromolecule from said derivative crystal.

The derivative crystal may be obtained by a crystallization process as described in the invention or by soaking a crystal from the biological macromolecule in a solution of a complex according to the invention or a cationic complex consisting of a lanthanide ion Ln³⁺ and a ligand of formula (I.1) to (I.5) as previously defined, as well as their salts with an anion, their solvates or hydrates. The derivative crystal may also be obtained by soaking a derivative crystal from the biological macromolecule obtained by a crystallization process as described in the invention, in a solution of a complex according to the invention or a cationic complex consisting of a lanthanide ion Ln³⁺ and a ligand of formula (I.1) to (I.5) as previously defined, or of a salt thereof with an anion, their solvates or hydrates.

In particular, the analysis of the crystalline structure of the biological macromolecule from said derived crystal corresponds to the determination of the structure of the biological macromolecule and is carried out by X-ray diffraction (RX). It is also possible that it corresponds to the obtaining of structural data by a high resolution method, such as X-ray diffraction (RX) or by a low resolution method by SAXS methods (Small-Angle X-ray Scattering) or MASC (Multiwavelength-Anomalous Solvent Contrast).

The following detailed description provides a better understanding of the invention.

The cationic complexes formed from a lanthanide ion Ln³⁺ and a ligand of formula (I), corresponding to complexes according to the invention or complexes formed with a ligand of formula (IA) to (IF) are cationic complexes, and in particular mono-cationic complexes. They are therefore capable of producing crystals derived from a biological macromolecule. Such derived crystals can be obtained by dipping or co-crystallization, as detailed below. The anomalous effect due to the presence of lanthanides in the derived crystals obtained leads to a phasing effect and facilitates the determination of the structure of the biological macromolecule concerned, in particular by X-ray diffraction. Complexes depending on the invention can therefore be used as an aid in determining or analyzing the crystalline structure of a biological macromolecule.

The cationic complexes formed from a lanthanide ion Ln³⁺ and a ligand of formula (I), corresponding to complexes according to the invention or complexes formed with a ligand of formula (IA) to (IF), as well as their salts, solvates and hydrates may also be used as an aid in the crystallization of biological macromolecules. Indeed, depending on their structure and the crystallization conditions used, the complexes according to the invention have at least one of the following properties:

-   -   nucleating effect: the presence of said lanthanide complex         during crystallization causes the growth of crystals under         conditions (buffer and/or additives and/or temperature and/or         concentration and/or duration . . . ) which do not allow the         formation of crystals in the absence of the complex according to         the invention.     -   Crystallizing effect: the presence of the said lanthanide         complex during tests of a series of crystallization conditions         (number of crystals and/or crystal size and/or improved         diffraction of the obtained crystals . . . ) allows to improve         the crystallization obtained, compared to a series of conditions         differing by the absence of complex. In the absence of         complexes, crystals appear but are of poorer quality.

In particular, the lanthanide complexes formed with ligands of formula (I) consisting of a lanthanide ion Ln³⁺ and a ligand of formula (I), corresponding to the complexes according to the invention or complexes formed with a ligand of formula (IA) to (IF), as well as their salts, solvates and hydrates, either increase the number of conditions under which the crystal formation of a macromolecule is possible, or to work under conditions lower in biological macromolecule and/or other precipitating agent conventionally used, either to improve the quality or the number of crystals thus favoring the obtaining of subsequent structural data, or to obtain several of these advantages.

The Europium and Terbium complexes also have the advantage of being luminescent under UV irradiation. Thus, their use allows an easy identification of the derived crystals during crystallization tests. This property can be used as a rapid detection method for crystallization platforms equipped with an automatic UV imaging device. In addition, this property can be used as an aid for centering in an X-ray beam during diffraction characterization, in particular.

Complexes depending on the invention may be prepared according to techniques adapted by the skilled person, techniques described in the examples or in application WO 2014/162105. The synthesis of complexes according to the invention is usually carried out in water. The complexes thus formed will therefore be more in the form of hydrate, but this complex can then be put in a different solvent, such as alcohol or DMSO, and give rise to a solvate or be dehydrated. In hydrated form, a complex according to the invention will contain up to 3 molecules of water per complex. On average, the number of water molecules per complex may, however, be different from an integer, e.g. equal to 0.5. For the applications envisaged in the context of the invention, lanthanide complexes depending on the invention may be used in hydrated form, or dehydrated, solvated or not, in powder form or in solution.

The quality and purity of a crystallizing agent are essential for the smooth implementation of the macromolecule crystallization when used as a crystallizing agent. Finally, lanthanide complexes according to the invention will be subjected to an appropriate purification step, in particular by steric exclusion chromatography.

The cationic complexes consisting of a lanthanide ion Ln³⁺ and a ligand of formula (I), corresponding to the complexes according to the invention or complexes formed with a ligand of formula (IA) to (IF), as well as their salts, solvates and hydrates, may be used as a crystallizing agent in any technique suitable for the crystallization of biological macromolecules.

For a detailed description of such techniques, reference can be made to the following reference works: <<Protein Crystallization, Second Edition (IUL Biotechnology Series)>> edited by Therese Bergfors or <<Crystallization of Nucleic Acids and Proteins: A Practical Approach>> edited by Arnaud Ducruix and Richard Giegé.

In particular, cationic complexes formed from a lanthanide ion Ln³⁺ and a ligand of formula (I), corresponding to complexes according to the invention or complexes formed with a ligand of formula (IA) to (IF), as well as their salts, solvates and hydrates, may be used as a crystallizing agent in a process for the crystallization of biological macromolecules by vapor diffusion, dialysis, batch or in a method of crystallization in cubic phase of lipids.

These different techniques will each be described briefly, the crystallizing agent being a lanthanide complex or a mixture of crystallizing agents containing at least one lanthanide complex with a ligand of formula (I) in the form of salts, solvates or hydrates:

-   -   Vapor diffusion crystallization is the most commonly used         crystallization method at present. This is the method generally         used in automated crystallization platforms. There are three         different schematic approaches in FIG. 2: the techniques of         sitting drop, hanging drop and sandwich drop. These three         techniques are shown schematically in FIG. 2.

In these three approaches, a solution of the crystallizing agent is placed in a well (light grey solution). The solution containing the biological macromolecule of interest is mixed with the crystallizing agent and deposited as a drop (either suspended, sitting or as a sandwich). The crystallization well is hermetically sealed with a glass plate or plastic film. At the start of crystallization, the concentration of the crystallizing agent in the drop is twice as low as in the well. Once the system is closed, a diffusion kinetics of the solvent from the drop (usually water) towards the well is set up. The volume of the drop will therefore decrease (loss of solvent i.e. of water) and the concentrations of biological macromolecule and crystallizing agent will thus increase, allowing, in favorable cases, to reach the nucleation phase. In this method, the concentration of crystallizing agent is extremely important. Indeed, an excess of crystallizing agent could lead to a diffusion kinetics of the solvent too fast and thus lead to the precipitation of the biological macromolecule. On the contrary, a too low concentration will not reach the nucleation zone. The concentration of the appropriate crystallizing agent will therefore be determined by routine tests carried out by the skilled worker. From the nucleation, crystalline growth will then decrease the concentration of soluble macromolecule until it reaches a stationary state.

-   -   Dialysis is another crystallization method, but it is less used         than steam diffusion. This technique is shown schematically in         FIG. 3.

The general principle is to slowly increase the concentration of the crystallizing agent. For this purpose, a solution containing the relevant biological macromolecule is placed in a dialysis container, e.g. capillary type or dialysis button closed by a semi-permeable membrane allowing the molecules of crystallizing agent (but not the macromolecule) to pass through. This container is then placed in a solution of crystallizing agent. An equilibrium between the solution containing the biological macromolecule of interest (which does not contain a crystallizing agent) and the external solution containing the crystallizing agent will be established. Thus, the concentration of the crystallizing agent in the dialysis container will increase and modify the solubility of the macromolecule. This technique has the advantage of being more precise than vapor diffusion with respect to maintaining pH and volume. However, the handling of crystals remains much more delicate.

-   -   Batch crystallization is the latest technique used to         crystallize biological macromolecules. This technique, like         dialysis, requires a large volume of solution.

The principle of this technique, shown schematically in FIG. 4, is simple: the highly concentrated macromolecule is mixed with the crystallizing agent. The oversaturated macromolecule solution will, over time, induce nuclei formation which will induce a decrease in the concentration of the macromolecule in solution, towards the formation of crystals.

The techniques of steam diffusion crystallization, crystallization by means of dialysis or batch crystallization work on all types of proteins, whether water-soluble or membranous, then soluble in detergent, as well as on other biological macromolecules (DNA, RNA, protein complexes, protein-DNA or RNA complexes, etc.).

For the crystallization of membrane proteins, it is also possible to use a lanthanide complex as a crystallizing agent in a technique of cubic phase lipid crystallization. The cubic phase crystallization of lipids of membrane proteins is described in the following publications: Landau et al. Lipidic cubic phase: A novel concept for the crystallization of membrane proteins, PNAS Vol. 93 no. 25 14532-14535 (1996), Caffrey et al. Crystallizing membrane proteins using lipidic mesophases. Nat Protoc 4 (5) 706-31 (2009), Cherezov V., Lipidic cubic phase technologies for membrane protein structural studies. Curr. Opin Struct Biol Vol 21 559-566 (2011).

Lanthanide complexes formed from a lanthanide ion Ln³⁺ and a ligand of formula (I), corresponding to complexes according to the invention or complexes formed with a ligand of formula (IA) to (IF), as well as their salts, solvates and hydrates are compatible with all crystallization methods which have just been detailed.

In particular, a solution containing a concentration of 1 to 100 mM, preferably 1 to 25 mM of a lanthanide complex formed from a lanthanide ion Ln³⁺ and a ligand of formula (I), corresponding to complexes according to the invention or complexes formed with a ligand of formula (IA) to (IF), in the form of a salt with an anion, optionally in the form of a solvate or hydrate and even more preferably 10 mM±10% will be used.

In crystallizations, in particular, in the vapor phase or batch, a solution of the biological macromolecule to be crystallized and a lanthanide complex consisting of a lanthanide ion Ln³⁺ and a ligand of formula (I), corresponding to the complexes according to the invention or complexes formed with a ligand of formula (IA) to (IF), in the form of a salt with an anion, optionally in the form of a solvate or hydrate (which, for simplification, will be referred to in the remainder of the description as a lanthanide complex), shall be prepared, preferably with a concentration of 1 to 100 mM, preferably 1 to 25 mM, and even more preferably at 10 mM±10%, as a lanthanide complex. The lanthanide complex formed from a lanthanide ion Ln³⁺ and a ligand of formula (I), corresponding to the complexes according to the invention or complexes formed with a ligand of formula (IA) to (IF), in the form of a salt with an anion, optionally in the form of a solvate or hydrate, can be directly solubilized by the solution containing the biological macromolecular to achieve a final complex concentration of 1 to 100 mM, preferably 1 to 25 mM, and preferably 10 mM±10% which corresponds to the concentration at which the nucleating effect is predominant. The lanthanide complex can also be introduced from a solution at a preferential concentration of 10 to 30 mM during the production of a crystallization drop: a volume of the macromolecular solution, an identical volume of the lanthanide complex solution and an identical volume of the well solution used for crystallization are then added successively.

In these crystallization techniques, the lanthanide complex and the biological macromolecule to be crystallized will generally be solved in water or in a buffered aqueous solution (see examples in the description of the crystallization kits in Annexes 2A and 2B), e.g. in an acetate, cacodylate, citrate, Bis tris propane, TRIS, HEPES, phosphate buffer solution. Solutions containing a biological macromolecule are usually buffered. If a solution of the lanthanide complex is formed, it will not necessarily be buffered. The solution containing the complex, the solution containing the biological macromolecule and/or the solution containing the biological macromolecule and the complex, depending on the crystallization technique used, will preferably have a pH in the range of 3 to 9.

Additives such as additives already known to act as precipitating/crystallizing agents or additives which initially facilitate the dissolution of the biological macromolecule to be crystallized may be added. Examples of these are those listed in the tables in Annex 2A or 2B, such as salts such as ammonium salts, sulphates (especially ammonium sulphate), acetates, phosphates, citrates (especially sodium citrate), formiates, tartrates such as sodium or potassium tartrate, double sodium and potassium tartrate, chlorides, iodine, and fluorides, e.g. Nacl; polymers such as polyethylene glycols and Jeffamine T; ethanol, dioxane, methylpentanediol, glycerol, isopropanol, 2-methyl-2,4-pentanediol. In particular, lanthanide complex can be used in any crystallization solution already on the market.

The concentration of macromolecule in the solution will theoretically be close to its solubility limit in the said solution, but may be more diluted, depending on the solution used.

Lanthanide complexes consisting of a lanthanide ion Ln³⁺ and a ligand of formula (I), corresponding to the complexes according to the invention or complexes formed with a ligand of formula (IA) to (IF), in the form of a salt with an anion, optionally in the form of a solvate or hydrate, may also be used to obtain derivative crystals from biological macromolecules, i.e. crystals containing said lanthanide complex. In particular, said lanthanide complex will be fixed on a specific position of the biological macromolecule to be studied, allowing the determination of the structure of said studied biological macromolecule.

The determination of the phases can be carried out with derived crystals obtained in a solution containing 1 to 100 mM of complex, preferably 10 to 100 mM, and in an even more preferable way from 100 mM±10% to the said lanthanide complex. However, in order to improve the quality of phasing, a soaking in a solution of a lanthanide complex, in particular, with a high concentration of complex, can also be carried out. This allows to increase the occupancy rate of the sites occupied by the complex.

Co-crystallization therefore involves adding the lanthanide complex during the crystallization process. Thus, during crystallization, the lanthanide complex can be inserted into specific crystalline sites leading to the production of derivative crystals.

In addition, to obtain a derivative crystal or increase the occupancy by a lanthanide complex of sites in the derivative crystal, a soaking of a biological macromolecule crystal in a lanthanide complex solution can be performed. It is possible to carry out a rapid soaking or long time soaking.

Rapid soaking consists of taking a crystal from a biological macromolecule of interest and soaking it briefly (notably from 45 seconds to 2 minutes) in a solution of the lanthanide complex. The solution in which the crystal is hardened has a high concentration of lanthanide complex, typically 50 to 500 mM, preferably 100 mM±10%.

In particular, a rapid soaking during the freezing phase of one or more crystals can be achieved. A rapid soaking during a freezing phase typically takes place in three steps: crystals are taken from their original drop and soaked in a soaking drop, corresponding to a solution equivalent to the original solution used for the formation of the said crystal(s) to which is added a concentration of 50 to 500 mM, preferably 100 mM±10% of lanthanide complex, and a cryoprotectant agent. After 45 seconds to 2 minutes, the crystal is soaked in a cryoprotective solution without lanthanide complexes to remove its excess. Finally, the crystal is immersed in liquid nitrogen and stored at low temperature, for example at 100K.

This soaking method is very efficient and allows to increase the occupancy rates and improve the quality of the phasing. For example, in the case of PhP1 protein, a 45-second soaking in a 100 mM complex solution increased the occupancy rate of the complex by 3. Rapid soaking prevents crystal from deteriorating and preserves its diffraction properties.

Long time soaking consists of taking a crystal from a biological macromolecule of interest and soaking it for a prolonged period of time (notably from 10 minutes to 24 hours) in a solution of the lanthanide complex. Advantageously, the solution in which the crystal is soaked has a high concentration of lanthanide complex, typically a concentration of 50 to 500 mM, preferably 100 mM±10%.

With the exception of duration, long time soaking can take place according to the same protocol as described for rapid soaking, especially during a freezing phase. However, the soaking drop is placed in equilibrium with a well containing the original solution used for the formation of the said crystal(s) in order to avoid dehydration of the drop.

Regardless of the type of soaking, it is possible to soak both crystals obtained by co-crystallization in the presence of a lanthanide complex and crystals obtained in the absence of such a complex (named native crystals in the context of this patent application). Soaking does not lead to any destruction or modification of the biological macromolecule. Lanthanide complexes allow to form derivative crystals from a biological macromolecule to obtain structural data for said biological macromolecule. In particular, structural data will be obtained from a derivative crystal from a biological macromolecule containing a lanthanide complex that acts as a phasing agent. These structural data can be obtained using a high-resolution method, such as the diffraction of X-rays (RX) or by a low resolution method, such as SAXS and MASC. Lanthanide complexes may be used as a phasing agent in X-ray diffraction (crystallization and structural analysis) or X-ray scattering, or as a contrast agent in SAXS or MASC, in electronic microscopy.

The use of lanthanide complexes consisting of a lanthanide ion Ln³⁺ and a ligand of formula (I) corresponding to the complexes according to the invention or complexes formed with a ligand of formula (IA) to (IF), in the form of a salt with an anion, optionally in the form of a solvate or hydrate, in particular as a phasing agent, has the advantage of not requiring a modification of the biological macromolecule by genetic engineering, as in the case of obtaining selenium proteins. There is no covalent binding between the lanthanide complex and the biological macromolecule of interest.

The following examples, with reference to the attached figures, illustrate the invention but are not specific.

FIG. 1 shows a phase diagram illustrating the crystallization process (extract from http://people.ds.cam.ac.uk/ml527/publications/assets/leunissen-literature-research.pdf)

FIG. 2 shows three different approaches: the techniques of sitting drop, hanging drop and sandwich drop.

FIG. 3 shows the dialysis technique: another crystallization method, which is still less used than vapor diffusion.

FIG. 4 schematically shows a batch crystallization technique used to crystallize biological macromolecules.

FIG. 5 shows phase diagrams of the lysozyme protein in absence (right) and in the presence of complex 10 or 17 determined at different crystallization times: the conditions, having produced crystals, are illustrated by black dots, the white dots being clear drops.

FIG. 6 shows phase diagrams of chicken breast white lysozyme in the absence and presence of complex 11 lysozyme determined after 4 days of crystallization. Crystal conditions are illustrated by black dots, white dots are clear drops and precipitate triangles.

FIG. 7 shows phase diagrams of the PB6 protein in the absence and presence of 10 mM of complex 10 determined after one day of crystallization. Crystal conditions are illustrated by black dots, white dots are clear drops and precipitate triangles.

FIG. 8 shows pb6 crystals (pb6-1 condition) in the presence of 10 mM complex 10 on the right (13% PEG—10 mg/ml) and without complex 10 on the left (15% PEG—20 mg/ml).

FIG. 9 shows the luminescence induced in the crystals by lanthanide complexes.

FIG. 10 shows a X-ray fluorescence spectrum measured on a 10 mM solution of complex 10 (left) and processed by the CHOOCH software (right). The wavelengths for recordings that allow optimal use of the anomalous diffusion of lanthanide (here the terbium) are indicated by an arrow.

FIG. 11 shows examples of experimental electron density maps.

FIG. 12 shows images obtained from the HTXIab crystallization robot.

EXAMPLES OF ACHIEVEMENTS

Part I: Synthesis and Characterization of Complexes

The following abbreviations are used:

Me=methyl; Moz=methoxyybenzyloxycarbonyl; Boc=tert-butoxycarbonyl; Ms=mesyl; Et=ethyl; TA=ambient temperatura; Ac=acetyl; DCM=dichloromethane; TACN=triazacyclononane; DMF=dimethylformamide; TFA=trifluoroacetic acid; ACN=acetonitrile; CCM=thin layer chromatography

Starting Materials and Characterization

All starting materials, solvents and salts of lanthanide were purchased from Sigma-Aldrich®, Acros Organics® and TCI® with purities greater than 98% for organic compounds and greater than 99.99% for lanthanide salts. These products were used directly without additional purification.

Chromatographs were carried out on neutral alumina activity III obtained by hydration of Acros Organics® alumina activity I (60Â) and on silica gel Acros Organics® (60 Å). The formed complexes have all been purified by Sephadex® steric exclusion column LH20.

The NMR spectra (¹H, ¹³C) were recorded on two Bruker® Advance devices operating at 500.10 MHz and 125.75 MHz for ¹H and ¹³C respectively, and at 300 MHz for ¹H for the second. Chemical displacements are partially reported per million (ppm) relative to the tetramethylsilane signature (¹H, ¹³C), with residual solvent peaks being used as internal reference.

The exact mass measurements were carried out at the Joint Centre for Mass Spectrometry (Villeurbanne, France).

A) Preparation of a First Batch of Triazacyclononane-Based Ligands/Complexes (TACH)

Compound 6 was prepared according to the procedure previously described in patent application WO2013/011236A1.

A1) Preparation of Compound 1

To a suspension of 20 g dipicolinic acid (0.12 mol, 1 eq.) in 120 mL methanol, 1 mL concentrated sulphuric acid is added. The mixture is carried in reflux for 24 hours. After cooling, the crystallized product is filtered and rinsed with cold methanol to give, after drying, 18 g of white crystalline powder of compound 1. (R=78%). ¹H-NMR (300 MHz, CDCl₃) δ (ppm)=8.26 (d, J=8 Hz, 2H), 7.99 (t, J=8 Hz, 1H), 3.98 (s, 6H).

A2) Preparation of Compound 2

18 g compound 1 (92 mmol, 1 eq.) are dissolved in 450 ml methanol and cooled to 0° C. 3.8 g NaBH₄ (101.2 mmol, 1.1 eq.) are then added. The mixture is then stirred 30 min at 0° C. and 30 min further, leaving the temperature slowly rising to RT. The reaction is stopped by adding 50 mL HCl (1M in H2O), then the organic phase is extracted with 100 mL dichloromethane. After washing the organic phase with brine (up to pH=7), drying with Na₂SO₄ and evaporation, the obtained product is purified by chromatography on silica gel (eluent: DCM/AcOEt 9/1 v/v up to pure AcOEt). We obtain 6 g of a white solid of compound 2 (R=40%). ¹H-NMR (300 MHz, CDCl₃) δ (ppm)=8.01 (d, J=8 Hz, 1H), 7.84 (t, J=8 Hz, 1H), 7.53 (d, J=8 Hz, 1H), 4.85 (d, J=3 Hz, 2H), 3.98 (s, 3H), 3.69 (bs, 1H).

A3) Preparation of Compound 3

3 mL of Et₃N (22 mmol, 3.2 eq.) is added to a solution of 1.2 g of alcohol 2 in 50 mL of dichloromethane at 0° C. and rapidly followed by 0.79 mL of mesyl chloride (10.2 mmol, I, 5 eq.). The mixture is then allowed to return at room temperature before heating for 1 hour at 50° C. Then 40 mL of water is added and the dichloromethane mixture (3×20 mL) is extracted. The obtained oily residue, after drying and evaporation of the organic phases, is finally purified on silica gel (eluent: CH₂Cl₂) to obtain a colorless oil of compound 3 which crystallizes in the freezer.

¹H NMR (500 MHz, CDCl₃) δ 8.12 (d, J=7.7 Hz, 1H), 7.93 (t, J=7.8 Hz, 1H, H10), 7.70 (d, J=7.8 Hz, 1H), 5.44 (s, 2H, H7), 4.01 (s, 3H, OMe), 3.15 (s, 3H, OMs).

¹³C NMR (126 MHz, CDCl₃) δ 165.33 (C13), 154.59 (s), 147.92 (s), 138.45 (C10), 125.41 (s), 125.13 (s), 71.11 (C7), 53.23 (OMe), 38.24 (OMs).

A4) Preparation of compound 6

Compound 6 is prepared by the method described above, (a) WO2013011236A1; b) 1.S. J. Butler, B. K. McMahon, R. Pal, D. Parker and J. W. Walton, Chem. Eur. J., 2013, 19, 9511-9517.)

A5) Preparation of compound 7

360 mg of compound 6 (1.19 mmol) and 760 mg of sodium carbonate (7.15 mmol, 6 eq.) are dried under reduced pressure in a schlenk before adding 100 mL of acetonitrile. Under argon, 710 mg of compound 3 (2.74 mmol, 23 eq.) are added to the suspension before heating for 12 h at 70° C. After return to room temperature, the mixture is filtered on sintered (porosity 4) and concentrated under reduced pressure. The product is purified by alumina chromatography (activity III, eluent DCM then DCM/MeOH 96/4 v/v) to finally obtain a yellow oil of compound 7 (532 mg, Yield: 80%).

¹H NMR (500 MHz, CDCl₃) δ 7.99 (dd, J=6 Hz, 2H, H11), 7.86-7.65 (m, 4H, H9), 3.98 (s, 10H, H7+OMe), 3.39 (d, J=30 Hz, 4H, H4H5), 3.11 (s, 2H, H3H6), 2.95 (s, 2H, H3′H6′), 2.66 (d, J=31 Hz, 4H, H1H2), 1.44 (s, 9H, boc).

¹³C NMR (126 MHz, CDCl₃) δ 166.01, 166.09 (C13), 161.43, 161.60 (C8), 155.88(CO(boc)) 147.45 (C12), 137.42 (d,C10), (s), 126.19, 126.46 (C9), 123.70 (C11), 79.48 (C(boc)), 63.70 (d, C7), 57.21 (C1C2), 55.37 (s), 55.11 (s), 54.67 (s), 53.04 (OMe), 50.54, 49.95 (C4C5), 28.72 (CH₃(boc)).

A6) Preparation of Compound 8

In 100 mL dichloromethane, 5 mL trifluoroacetic acid is added to a 427 mg solution of compound 7. After 5 hours of agitation at room temperature, the mixture is evaporated by removing the TFA by drive with several toluene additions. The resulting residue is purified by alumina chromatography (activity III, eluent DCM/MeOH (gradient 1% to 7%)). The viscous yellowish solid obtained from compound 8 is stored under argon in the freezer (mass: 315 mg, yield: 90%).

¹H NMR (500 MHz, CDCl₃) δ 7.96 (dd, J=7.7, 0.5 Hz, 2H, H11), 7.71 (t, J=8 Hz, 2H, H10), 7.42 (dd, J=7.9, 0.5 Hz, 2H), 3.98 (d, 10H, H7+OMe), 3.40 (t, J=5 Hz, 4H, H4H5), 3.03 (t, J=5 Hz, 4H, H3H6), 2.71 (s, 4H, H1H2).

¹³C NMR (126 MHz, CDCl₃) δ 65.37 (C13), 159.43 (C8), 147.59 (C12), 137.87 (C10), 126.10 (C9), 124.02 (C11), 60.95 (C7), 54.60 (C1C2), 53.31 (OMe), 51.66 (C3C6), 46.52 (C4C5).

NB: the synthesis of this compound has been previously described in the following reference: A. Nonat, C. Gateau, P. H. Fries, M. Mazzanti, Chem. Eur. J., 2006, 12, 7133.

A7) Preparation of Compound 9

Ligand 9 is generated in situ by saponification of diester 8 in the presence of sodium carbonate Na₂CO₃ (2 eq.) in a MeOH/H20 mixture (1/1, v/v) stirred for 3 hours at 50° C.

¹H NMR (500 MHz, D₂O) δ 7.77 (d, J=7.1 Hz, 2H, H9), 7.69 (t, J=7.7 Hz, 2H, H10), 7.29 (d, J=7.2 Hz, 2H, H11), 3.86 (s, 4H, H7), 3.08 (t, J=5.9 Hz, 4H, H4H5), 2.91 (t, J=5.9 Hz, 4H, H3H6), 2.65 (s, 4H, H1H2).

¹³C NMR (126 MHz, D₂O) δ 173.22 (C13), 158.44 (C8), 153.01 (C12), 138.23 (C10), 125.17 (C11), 122.40 (C9), 60.27 (C7), 50.53 (C1,C2), 47.12 (C3,C6), 44.10 (C4,C5).

A8) Preparation of Compound 10

After reaction for 3 h at 50° C. of 110 mg diester 8 (0.26 mmol) with 83 mg Na₂CO₃ (0.78 mmol, 3.0 eq.) and neutralization by HCl (1M), the disappearance of the ester is verified by NMR. 96 mg terbium chloride (0.26 mmol, 1 eq.) are added to compound 9, formed in situ, before stirring the mixture for 12 hours at 50° C. Solubilized in a minimum of methanol, complex 10 is then separated from the various salts by centrifugation. The last traces of salts are removed by passing through a steric exclusion column (LH20, Sephadex®, eluent: water). MS (ESI-TOF) calculated M+: 556.1003; measured: 556.0990

A9) Preparation of Compound 11

An identical protocol to that/the one used for the preparation of Complex 10 is used with 170 mg diester 8 (0.4 mmol) and 219 mg EuCl3,6H20 (0.6 mmol, 1.5 eq.). MS (ESI-TOF) calculated M+: 550.0962; measured: 550.0957.

B) Preparation of a Second Series of Triazacyclononane-Based Ligands (TACN)

B1) Preparation of Compound 12

15 g of monohydric chelidamic acid (0.075 mol, I eq.) are dissolved in 120 mL of thionyl chloride under argon. The suspension is cooled to 0° C. and 3 mL DMF are added. The reaction mixture is then stirred for 12 hours with reflux. Volatiles are evaporated after several toluene additions to remove the last traces of SOCl₂. The resulting yellowish solid is then dissolved in methanol and the mixture is refluxed for 12 hours to complete the reaction. After evaporation of the solvent, the residue is taken up by dichloromethane and washed successively with a saturated solution of NaHCO₃, water and brine. The organic phase is then dried on Na₂SO₄, filtered and evaporated. Pure compound 12 is obtained by recrystallization in methanol to obtain 8.3 g of white crystalline powder. (R=44%).

¹H-NMR (300 MHz, CDCl₃) δ (ppm)=8.29 (s, 2H), 4.03 (s, 6H).

B2) Preparation of Compound 13

6 g of compound 12 (0.026 mol, I eq.) are dissolved in 250 mL of a DCM/methanol mixture (150/100, v/v) and the solution is cooled to 0° C. Then, 1.09 g of NaBH₄ (0.029 mol, 1.1 eq.) are added in one step before stirring the mixture for 30 min at 0° C. and 30 min at RT. The reaction is stopped by the addition of 50 mL hydrochloric acid (1M) and 100 mL of water. The organic phase is then washed with water to a pH=7, then washed in brine, dried on Na₂SO₄ and evaporated. The reaction crude is then purified on silica gel (eluent: DCM/acOEt 1/1 v/v) to obtain after evaporation 1.5 g of a white powder of compound 13 (R=30%).

¹H-NMR (300 MHz, CDCl₃) δ (ppm)=8.00 (bd, 1H), 7.60 (bd, 1H), 4.85 (d, J=7 Hz, 2H), 3.99 (s, 3H) 3.48 (t, J=7 Hz, 1H).

B3) Preparation of Compound 14

1.5 g of compound 13 (7.4 mmol, 1 eq.) are dissolved in 200 mL dichloromethane and 3 mL triethylamine are added (22.2 mmol, 3 eq.). Then 0.87 mL of mesyl chloride is added (11.1 mmol, 1.5 eq.) slowly and a progressive yellow coloring of the mixture is observed. The reaction progress is followed by CCM and stopped after 30 min. 100 mL of a saturated solution of NaHCO₄ are added and the organic phase is washed with water (up to pH=7) and brine. The organic solution is then dried on Na2S04 and evaporated to give 2 g of a yellow oil of compound 14 used without further purification (quantitative yield).

¹H-NMR (300 MHz, CDCl₃) δ (ppm)=8.1 (s, 1H), 7.68 (s, 1H), 5.40 (s, 2H), 4.01 (s, 3H), 3.17 (s, 3H).

B4) Preparation of Compound 15

200 mg of triazacyclononane mono-boc (0.7 mmol, I eq.) are suspended in 100 mL of dry acetonitrile with 420 mg of anhydrous sodium carbonate. 463 mg of compound 3.3 (1.75 mmol, 2.5 eq.) are then added. After cooling, the mixture is sintered to remove the carbonate before evaporation of the solvent. The residue is taken up in dichloromethane and purified by alumina column chromatography (activity III, eluent: dichloromethane and ethyl acetate). 250 mg of a yellow solid of compound 15 (R=63%).

¹H-NMR (300 MHz, CDCl₃) δ (ppm)=8.00 ppm (d, J=1 Hz, 1H), 7.99 (d, J=1 Hz, 1H), 7.89 (d, J=1 Hz, 1H), 7.72 (d, J=1 Hz, 1H), 3.99 (m, 10H), 3.39 (m, 4H), 3.03 (m, 4H), 2.67 (m, 4H), 1.48 (s, 9H). LC-MS: [M+H]⁺=596.2 m/z.

B5) Preparation of Compound 16

3 mL trifluoroacetic acid is added to a 110 mg solution of compound 15 in 60 mL of dichloromethane, The mixture is then stirred for 12 hours at room temperature. The solvent is then evaporated and the TFA is evaporated by several additions of a methanol/toluene mixture. The residue is then taken up by 50 mL of dichloromethane and washed with water to pH=7. The organic phase is then dried on Na₂SO₂ and evaporated to obtain 100 mg of white powder of compound 16. (R−95%).

¹H-NMR (300 MHz, CDCl₃) δ (ppm)=7.97 (d, J=1 Hz, 2H), 7.54 (d, 7=1 Hz, 2H), 4.04 (s, 6H), 4.03 (s, 4H), 3.35 (m, 4H), 3.00 (m, 4H), 2.76 (s, 4H).

¹³C-NMR (75 MHz, CDCl₃) δ (ppm)=163.91, 160 (q, J=40 Hz), 147.58, 146.99, 126.49, 125.22, 60.37, 53.80, 53.56, 53.28, 45.39.

HR-MS: [M+H]⁺=496.1501 m/z, theor. for C₂₂H₂₈Cl₂N₄O₄ is 496.1513 m/z.

B6) Preparation of Compound 17

100 mg of compound 16 (0.2 mmol, 1 eq.) are suspended in 30 mL of water and 32 mg of sodium hydroxide (0.81 mmol, 4 eq.) are added. The mixture is then stirred at 60° C. during one hour. The complete hydrolysis of the ester functions is confirmed by LC-MS. The solution is then cooled and its pH is adjusted to 5 with a progressive addition of a hydrochloric acid solution (1M). 5 mL of methanol is then added before introducing 42 mg of sodium carbonate to the solution. 91 mg of TbCl₃, 6H₂O are added before stirring the mixture at 50° C. for 12 hours. After return to ambient temperature, the insoluble salts are filtered on sintered glass before evaporation of the solvents to obtain 300 mg of raw product. The complex is purified by steric exclusion chromatography (Sephadex® LH20, eluent: water) to finally obtain 55 mg of a colorless crystalline product (yield=44%).

¹H-NMR (300 MHz, D₂O) δ (ppm)=121.1, 83.90, 69.15, 52.71, 46.23, 29.58, 26.06, −0.96, −11.39, −20.2, −33.57, −36.26, −44.20, −70.35, −91.19, −92.47, −120.84.

HR-MS: M⁺=624.0213 m/z, theor. for C₂₀H₂₁Cl₂N₅O₄Tb 624.0219 m/z.

C) Preparation of a Third Series of Triazacyclononane-Based Ligands (TACN)

C1) Preparation of Compound 19

8.3 g of compound 12 (36 mmol, 1 eq.) are dissolved in 200 mL acetonitrile and 54 g sodium iodide are added (0.36 me, 10 eq.) and 10 mL acetyl chloride (0.108 mol, 3 eq.). The suspension is then placed in an ultrasonic bath for 3 hours. Then 200 mL of dichloromethane are added and the organic phase is washed with a saturated solution of sodium hydrogen carbonate. The organic phase is then washed with water up to pH=7, dried on sodium sulfate and evaporated to obtain 10.7 g of off-white solid of compound 19, used without further purification (R=92%).

¹H-NMR (300 MHz, CDCl₃) δ (ppm)=8.65 (s, 2H), 4.02 (s, 6H).

C2) Preparation of Compound 20

1.39 g sodium borohydride (36.6 mol, 1.1 eq.) are added to a solution of 10.7 g (33.3 mmol, 1 eq.) of compound 19 in 200 ml of a mixture of methanol/dichloromethane (140/60) and cooled to 0° C. The mixture is then stirred for 30 min at 0° C. and 30 min at room temperature. The reaction is then stopped by adding 50 mL of an hydrochloric acid solution (1M). The organic phase is then washed with water up to pH=7, dried on Na₂SO₄ and evaporated. The raw product is then purified on silica gel (eluent: dichloromethane with progressive addition of methanol (0 to 10%)) to obtain 5 g of a white powder of compound 20. (R=51%).

¹H-NMR (300 MHz, CDCl₃) δ (ppm)=8.39 (bd, 1H), 7.97 (bd, 1H), 4.82 (d, J=5 Hz, 2H), 3.99 (s, 3H) 3.04 (t, J=7 Hz, 1H).

C3) Preparation of Compound 21

To a solution of 1 g of compound 20 (3.4 mmol, 1 eq.) and 1.4 mL of triethylamine (10.2 mmol, 3 eq.) in 120 mL of dichloromethane are added 0.4 mL of mesyl chloride (5.1 mmol, 1.5 eq.). After 30 minutes of stirring (reaction followed by TLC), 100 mL of a saturated solution of NaHC03 is added. The organic phase is then washed with water, dried on Na₂SO₄ and evaporated. 1.1 g of yellow oil of compound 21 is obtained and used without further purification (R=95%).

¹H-NMR (300 MHz, CDCl₃) δ (ppm)=8.46 (s, 1H), 8.04 (s, 1H), 5.36 (s, 2H), 4.00 (s, 3H), 3.16 (s, 3H).

C4) Preparation of Compound 22

60 mg of triazacyclononane mono-boc 6 (0.2 mmol, 1 eq.) are suspended in 50 mL of dry acetonitrile under argon with 140 mg of anhydrous Na₂CO₃ (1.2 mmol, 6 eq.). A solution of 184 mg of compound 21 (0.5 mmol, 2.5 eq.) in dry acetonitrile is then added and the mixture is stirred for 12 hours at 60° C. under argon. After cooling, the mixture is filtered on sintered and evaporated. The residue is taken up by dichloromethane and purified on an alumina column (activity III; eluent: dichloromethane followed by ethyl acetate) to obtain 110 mg of pure compound 22 in the form of a white pasty solid (R=71%). ¹H-NMR (300 MHz, CDCl₃) δ (ppm)=8.34 (s, 2H), 8.22 (s, 1H), 8.11 (s, 1H), 3.97 (s, 6H), 3.94 (s, 4H), 3.4-2.58 (m, 12H), 1.47 (s, 9H).

¹³C-NMR (75 MHz, CDCl₃) δ (ppm)=164.6, 162 (d, J=14 Hz), 155.61, 147.6 (d, J=14 Hz), 135.3 (d, J=14 Hz), 132.7, 106.6, 79.5, 63.1, 56.6, 54.9, 54.4, 53.1, 50.8, 50.2, 28.7. LC-MS: [M+H]⁺=780.0 m/z.

C5) Preparation of Compound 23

110 mg of compound 22 (0.14 mmol, 1 eq.) are dissolved in 50 mL of dichloromethane, then an excess of trifluoroacetic acid is added (3 mL). The mixture is then stirred at room temperature for 12 hours. The solvent is then evaporated and a mixture of 20 mL of dichloromethane and 10 mL of water is added. The aqueous phase is neutralized by adding a saturated solution of NaHCO₃ and extracted with dichloromethane. All the organic phases are dried on sodium sulfate and evaporated. The result is 100 mg of white solid (quantitative yield) of compound 23, which is used without further purification.

⁷¹H-NMR (300 MHz, CDCl₃) δ (ppm)=8.23 (s, 2H), 7.83 (s, 2H), 3.92 (s, 10H), 3.27 (s, 4H), 2.92 (s, 4H), 2.67 (s, 4H). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm)=164, 159.98, 147.81, 134.86, 133.14, 107.08, 59.85, 5336, 52.65, 49.87, 45.33. HR-MS: [M+H]⁺=680 m/z, theor. for C₂₂I₂H₂₈N₂O₅ 680.0225 m/z.

C6) Preparation of Compound 24

100 mg of compound 23 (0.15 mmol, 1 eq.) are suspended in 30 mL of water and 24 mg of sodium hydroxide (0.81 mmol, 4 eq.) are added. The mixture is then stirred at 60° C. for one hour. The complete hydrolysis of the ester functions is confirmed by LC-MS. The solution is then cooled, and its pH is adjusted to 5 with a progressive addition of a hydrochloric acid solution (1M). 5 mL of methanol are then added before introducing 42 mg of sodium carbonate to the solution. 66 mg of TbCl₃, 6H₂O (0.18 mmol, 1.2 eq) are added before stirring the mixture at 50° C. for 12 hours, after return to ambient temperature, the insoluble salts are filtered on sintered glass before evaporation of the solvents to obtain 200 mg of raw product. The complex is purified by steric exclusion chromatography (Sephadex® LH20, eluent: water) to obtain 36 mg of a colorless crystalline product (30% yield).

¹H-NMR (300 MHz, D₂O) δ (ppm)=122.8, 84.95, 82.17, 70.62, 49.22, 25.03, 20.8, −0.89, −8.54, −19.41, −33.05, −41.19, −67.6, −88.49, −90.57, −125.1.

HR-MS: [M+H]⁺=807.8921 m/z, theor. for C₂₀H₂₁I₂N₅O₄Tb 807.8931 m/z.

D) Preparation of a Fourth Series of Triazacyclononane-Based Ligands (TACH)

D1) Preparation of Compound 25

1 g of compound 2 (6 mmol, 1 eq.) is dissolved in 100 mL methanol and 8 mL of a 30% aqueous ammonia solution (60 mmol, 10 eq.). This mixture is stirred at room temperature for 12 hours. The solvents are then evaporated to give 900 mg of a white solid of the pure compound 25 (=quantitative).

¹H-NMR (300 MHz, C₂D₆SO) δ (ppm)=8.14 (bs, 1H), 7.96 (t, J=8 Hz, 1H), 7.88 (d, J=8 Hz, 1H), 7.61 (bd, J=8 Hz, 2H), 4.64 (s, 2H).

LC-MS: [M+H]⁺=153.2 m/z.

D2) Preparation of Compound 26

To 1 g of compound 25 (6.6 mmol, 1 eq.) in 20 mL DMF at 0° C., 4 mL thionyl chloride (53 mmol, 8 eq.) are added. The mixture is stirred for 2 hours, before being allowed to return to room temperature in 15 minutes. 150 mL water is then added before extracting the product with 30 mL dichloromethane. The organic phase is then washed with water to pH=7, washed in brine, dried on Na₂SO₄ and evaporated. The resulting residue is purified by silica gel chromatography (eluent: dichloromethane) and leads to the production of 720 mg of a white solid of compound 26 (R=55%).

¹H-NMR (300 MHz, CDCl₃) δ (ppm)=7.89 (t, J=8 Hz, 1H), 7.75 (d, J=8 Hz, 1H), 7.65 (d, J=8 Hz, 1H), 4.69 (s, 2H).

LC-MS: [M+H]⁺=153.3.

D3) Preparation of Compound 27

120 mg of triazacyclononane protected by a mono-Boc (0.4 mmol, 1 eq.) are dissolved in 50 mL of dry acetonitrile under argon with 252 mg of anhydrous anhydrous Na₂CO₃ (2.4 mmol, 6 eq.). A 151 mg solution of compound 26 (1 mmol, 2.5 eq.) in dry acetonitrile is then added and the mixture is stirred for 12 hours at 60° C. under argon. After cooling, the mixture is filtered on sintered and evaporated. The residue is taken up by dichloromethane and purified on an alumina column (activity III; eluent: dichloromethane followed by ethyl acetate) to give 160 mg of compound 27 in the form of a white pasty solid (R=83%).

¹H-NMR (300 MHz, CDCl₃) δ (ppm)=7.79 (dt, ³J=8 Hz, ⁴J=1 Hz, 2H), 7.70 (bd, J=8 Hz, 2H), 7.57 (dt, ³J=8 Hz, ⁴J=1 Hz, 2H), 3.91 (s, 4H), 3.36 (m, 4H), 3.03 (m, 4H), 2.66 (m, 4H), 1.47 (s, 9H).

LC-MS: [M+H]⁺=462.2 m/z.

D4) Preparation of Compound 28

225 mg of NaN₃ (3.5 mmol, 10 eq.) and 185 mg of dry NH₄Cl are added to a 160 mg solution of compound 27 (0.35 mmol, 1 eq.) in 40 mL of dry DMF under argon. The mixture is stirred at 120° C. for 12 hours. After cooling, sintered filtration and evaporation of the solvents, a residue is obtained, which is taken up by 50 mL hydrochloric acid (1M) and placed in an ultrasonic bath for 2 hours.HR-MS [M+H+]: 604.1439 th 604.1447

D5) Preparation of Compound 29

155 mg of compound 28 (0.35 mmol, 1 eq.) are suspended in 10 mL of water with 220 mg Na₂CO₃ (2.08 mmol, 6 eq.). After 10 min of stirring, 155 mg TbCl₃*6H₂O (0.42 mmol, 1.2 eq.) are added. The solution is then stirred for 12 hours at room temperature. After evaporation of solvents, the raw product is purified on a steric exclusion column. (Sephadex® LH20 in water) to finally obtain 40 mg of crystalline white solid (yield=20%).

HR-MS: [M+H]⁺=604.1439 m/z, theor. for C₂₀H₂₃N₁₃Tb 604.1427 m/z.

E) Preparation of a First Series of Ligands Based on 1,7-dioxa-4,10-Diazacyclododecane

Compound 31 is prepared according to the method previously reported (M. Mato-Iglesias, Adrián Roca-Sabio, Z. Pálinkás, D. Esteban-Gómez, C. Platas-Iglesias, E. Tóth, A. de Blas, T. Rodríguez-Blas, Inorg. Chem., 2008, 47, 7840).

Part II: Crystallographic Studies

A) Model Proteins Studied

5 proteins, of which three commercial proteins were selected. The structure of these five proteins was known. These five proteins were chosen because of their physico-chemical differences. They belong to various organisms, have broad thermal properties and an oligomeric state ranging from monomer to hexamer. Tests with proteins of unknown structure were also carried out.

a. Commercial Proteins

The three commercial proteins selected are chicken's egg white lysozyme (HEWL), Thaumatococcus danielli Thaumatin and Tritirachium album Proteinase K. These three proteins are model proteins for crystallogenesis. They are purchased under freeze-dried form. They were solubilized in milliQ water just prior to crystallogenesis experiments at the desired concentration. These are presented in Table 1 below.

TABLE 1 Commercial proteins used and associate supplier Number of Supplier product Proteins amino acids reference Sequence HEWL 129 10 837 059 001 SEQ ID No 1 Roche Thaumatine 207 T7638 SEQ ID No 2 Sigma Proteinase K 384 03 115 879 001 SEQ ID No 3 Roche

The conditions of native crystallization (i.e., without addition of complexes depending on the invention) of these proteins are known and described in Table 2 below. These conditions were used to characterize the effect of lanthanide complexes on the crystallogenesis of these commercial proteins.

TABLE 2 Usual crystallization conditions for commercial proteins Proteins Buffer Salt HEWL 100 mM sodium 0; 5 to 2M NaCl acetate pH 4.6 Thaumatine 100 mM Bis tris 0.9 to 1.4M Tartrate propane pH 6.5 twice of Na+ and K+ Proteinase K 100 mM sodium 0.9 to 1.5M cacodylate pH 6.5 ammonium sulfate

a. Non-Commercial Proteins

The two non-commercial proteins (known structures) are Pyrococcus horikoshii Protease 1 and Pyrococcus furiosus reductase Glyoxylate hydroxypyruvate reductase. These two proteins have been purified according to the protocols described in the references below:

-   -   Protease 1 protein purification protocol of P. horikoshii:         Xinlin Du et al. Crystal structure of an intracellular protease         from Pyrococcus horikoshii at 2-A resolution. 2000. Flight 97.         N° 26. PNAS.     -   Protocol for purification of P. furiosus Glyoxylate reductase         protein: Thesis of Louise Lassalle, defended on 19 Dec. 2014 in         Grenoble: Molecular bases of piezophilic adaptation: structural         and biochemical studies of key enzymes of metabolism coming from         archaeas and bacteria isolated in the seabed. Link:         http://www.theses.fr/s98711.

The technical details concerning these proteins are given in Table 3 below:

TABLE 3 Characteristics of non-commercial proteins used Size (amino Physico-chemical Link to Biological Name Organism Vector acids) characteristic sequence unit GRHPR P. furiosus pET41 336 Hyperthermophilic 1 dimeric Protease 1 P. horikoshii pET41c 166 Thermophilic 2 hexameric Sequence of GRHPR: SEQ ID No 4 Sequence of protease 1: SEQ ID No 5

The crystallization conditions for these two proteins are described in Table 4 below. These conditions allow the proteins to crystallize in their native form and are extracted from the same references as purification protocols.

TABLE 4 Crystallization conditions of the non-commercial proteins used Proteins Buffer Salt Precipitating agent GRHPR 100 mM sodium 100 mM NaCl 14 to 24% acetate pH 5.2 Protease 1 PEG 400

The five proteins described above are the reference proteins for characterizing the effects of lanthanide complexes.

c. Unknown Proteins

The specificities of the three proteins with unknown structure are described in Table 5 below. The structure of the MDH-ANC80 protein was determined using the lanthanide 17 complex. The individual steps will be described individually in a separate section.

TABLE 5 Characteristics of proteins of unknown structure on which lanthanide complexes according to the invention have been tested Name Organism Vector Size AA characteristics Name Organism pb6 Phage T5 pET41 464 Viral protein 1 monomer MDH-ANC80 The sequence pET41c 309 Halophilic 2 Tetramer was generated bio-informatically Sequence of Pb6: SEQ ID No 6 Sequence of MDH: SEQ ID No 7 No crystallisation conditions were published for these two proteins. Sequence SEQ ID N° 1 to 7 are presented in ANNEXE1.

The ANC80 Malate Dehydrogenase (MDH) purification protocol is described in the literature (Madern et al. 1995 230 (3): 1088-95 Eur. J. Biochem).

The protocol for the purification of pb6 was established by Cécile Breyton's team from the M&P group of the Institut de Biologie Structurale and is as follows:

Pb6 Purification Protocol:

Expression system=E. coli BL21 (DE3) transformed with LIM1 (Kan R) His tag with cleavage site “Tobacco Etch Virus”.

-   -   Preculture medium LB classic medium with 50 g/ml of kanamicyne     -   Classical culture in LB medium: Seeding with an optical density         of 0.1 and 50 μg/ml of Kanamicyne.     -   After 6 hours of culture, centrifugation 30 minutes 4000 rpm     -   Bacteria freezing at −80° C.     -   Breakage     -   Recovery in lysis buffer 50 mM Tris pH 8, 150 mM NaCl, 2 mM         MgCl2 in the presence of antiprotease and DNase     -   Microfluidizer Bacteria Lysis 6×12000 psi     -   Centrifugation: 20 minutes at 14,000 rpm     -   Nickel affinity column

Balancing buffer=20 mM Tris pH 8, 250 mM NaCl, 15 mM Imidazole

Elution buffer—20 mM Tris pH 8, 200 mM Imidazole

Flow rate=1 ml/min

-   -   Dilution of the elution fractions to the fifth with distilled         water to reduce conductivity.     -   Ion exchange column (HT Q 1 ml)

Balancing buffer=20 mM Tris pH 8

Elution buffer=20 mM Tris pH 8 1M NaCl

Emission by linear gradient

Flow rate=1 ml/min

-   -   Concentrator concentrate with 30 kDa diaphragm     -   Desalination by gel filtration in a pH 8 20 Mm sorting buffer

B) Automated Crystallization/Stability of Lanthanide Complexes

a. The HTXIab Platform.

To determine the crystallization conditions of a protein, the HTXIab platform was used to screen a wide range of crystallization conditions. These conditions are all described in the Tables in Appendix 2A and 2B and derived from https://embl.fr/htxlab/index.php?option=com_content&view=article&id=38&Itemid=172.

A conventional screening consists of 6 crystallization plates of 96 wells each, representing 576 conditions.

The studies conducted on MDH-ANC80 and Pb6 used the conditions described in Appendix 2A.

The studies carried out on the other proteins were carried out under the conditions described in Appendix 2B, due to a change due to the supplier.

Complex Stability

The stability studies were performed under the conditions described in Appendix 2A. Lanthanide complexes based on tris-dipicolinate (DPA) [Ln(DPA)₃]³⁻ have been shown to exhibit” self-crystallization “for certain crystallization conditions. In particular, the presence of divalent cations, high salt concentrations, the presence of MPD caused the self-crystallization of this type of complex (Doctoral thesis of R. Talon defended in Grenoble on Jun. 6, 2012). Two concentrations of Ln(DPA)₃ ³⁻ have been evaluated (25 and 100 mM). Thus, out of 576 conditions, more than a quarter of the conditions lead to self-crystallization/precipitate formation of Ln(DPA)₃ ³⁻ at 100 mM and of the order of 8% when used at 25 mM.

Equally, the stability of complex 10 was evaluated at 3 different concentrations (10, 50 and 100 mM). Complex 10, even at a concentration of 100 mM, exhibits increased stability. Indeed, no crystal of the complex was observed, as detailed below where only precipitates were detected.

Summary of the screening of the 6 classical plates for 100 mM of complex 10:

Plaque Hampton 3

Slight precipitation observed in the presence of NaHPO₄ or KHPO₄.

Plaque Hampton 5

Mostly PEGs in this case 100% of the drops are clear.

Plaque Hampton 4

Mostly salts. Some conditions are redundant with the Hampton 3 plate. Similar effect in the presence of HPO₄ ⁻ with slight observed precipitation.

Plaque Qiagen 1

Mixture of PEGs, salts and metals. A slight precipitate is observed in the presence of cadmium acetate.

Plaque Hampton 6

Slight precipitate observed in the presence of a mixture of metals (cadmium, zinc, cobalt).

Plaque Hampton 2

Slight precipitate in the presence of NaF and (NH₄)₂PO₄

Below is a detailed description of the conditions that lead to these precipitates: With 100 mM of Complex 10 added to the standard conditions:

Hampton Plate 4: 21 precipitated conditions No. A7, B7, C7, C7, A8, B8, ABCD 9, 10, 11 and 12 (mainly ammonium phosphate)

Hampton Plate 5: No precipitate

Quiagen Plate 1: 6 precipitated conditions No. D6, B9, F9, F9, F9, D10, E10

Hampton 6 plate: 4 precipitated conditions No. A3, B3, C3, H8

Hampton Plate 2: 7 precipitated conditions No. C2, C5, B6, A7, C7, C7, A12, B12

Hampton Plate 3: 4 precipitated conditions No. H3, E4, A6, E5

This represents a total of 42 precipitated conditions. On a screening of 576 conditions this is equivalent to 7.3%;

No false positives like with DPA.

With 10 mM of complex 10 added to the standard conditions:

Hampton Plate 4: No precipitate marked as observed with some 100 mM, some trace of precipitate for ABCD line 12

Qiagen Plate 1: a slightly precipitated condition for E10

Wizard plate I and II rigaku 5 precipitated conditions No. E6, C7, B9, C10, H11

JCSG plate: 3 precipitated conditions No. D2 G5, A6

PACT Qiagen Plate: 5 precipitated conditions No. E1, A5, A6, E11, C12

PEGs Qiagen Plate: No precipitated condition

A total of 18 conditions with slight traces of precipitation. (3.2% of the screening) considering that drops with some traces of precipitate were described as “precipitated”. The amount and number of precipitating assays have nothing to do with the observed precipitates at 100 mM of complex 10, nor at 25 mM of (DPA)₃ ³⁻.

It should be noted that the presence of these precipitates is not incompatible with the appearance of protein crystals. In fact, manual crystallization tests of the C. aurantiacus MDH protein in the presence of cadmium have made it possible to obtain crystals in the presence of 50 mM of complex 10.

C) Screening of New Crystallization Conditions

To carry out a screening of six plates at the HTXIab platform, 100 μl of protein solution are required. In order to have a direct comparison, the native protein (without lanthanide complex) and the protein in the presence of 10 mM complex 10 were screened in parallel in the same crystallization plates.

a. Protocol for the Preparation of Protein Samples Containing Lanthanide Complex.

Lanthanide complexes were stored under powder form at 4° C. The mass corresponding to 10 mM per 100 μl of protein solution was weighed on a precision scale. The powder was centrifuged to form a pellet. This pellet was taken up by 100 μl of the protein solution. A two-minute centrifugation was carried out to eliminate any aggregates. The solution was then changed to a new tube. This protocol has been applied for all proteins sent to the robot.

b. Determination of New Conditions for Unknown Protein pb6

The most interesting conditions in terms of crystallogenesis and allowing the crystallization of pb6 are listed in Table 6. The conditions under which crystals were formed only in the presence of 10 mM of complex 10 are indicated in bold (they are pb6-I and pb6-4).

TABLE 6 Crystallization conditions for pb6 protein obtained in the HTX robot in the absence or presence of complex 10 Native/ Condition complex Precipitating reference 10 Buffer Salt agent pb6-1 10 mM 0.1M HEPES 0 10% PEG 6K complex 10 pH 7 pb6-2 Native 0.1M sodium 0  8% PEG 4K acetate pH 4.6 pb6-3 Native 0.1M MES pH 6 0 15% PEG 5KMME pb6-4 10 mM 0.1M Bis tris 0.2M 45% MPD complex 10 pH 6.5 ammonium acetate

The pb6-I condition was reproduced manually in the laboratory and also gave crystals. The use of complexes according to the invention makes it possible to double the number of conditions that led to crystallization.

c. Determination of New Conditions for Unknown Protein MDH-ANC80

The most interesting conditions in terms of crystallogenesis and allowing the crystallization of protein ANC80 are listed in Table 7. The conditions which allowed the crystals to be generated only in the presence of 10 mM of complex 10 are mentioned in bold (they are ANC-1 and ANC-2).

TABLE 7 Crystallization conditions for the ANC80 protein obtained at the HTX robot in absence or in the presence of complex 10 Condition Native/complex Precipitating reference 10 Buffer Salt agent ANC-1 10 mM 0.1M MES 0 65% MPD complex 10 pH 6   ANC-2 10 mM 0.1M HEPES 0.2M MgCl₂ 30% PEG 400 complex 10 pH 7.5 ANC-3 Native 0.1M MES 0.01M zinc 25% PEG pH 6.5 sulfate 550MME ANC-4 Native and 10 0.1M MES 1M lithium PEG 6K mM complex 10 pH 6   chloride 30%

The ANC-1 condition was replicated in the laboratory and allowed crystal growth (Paragraph D. a). The use of complexes according to the invention makes it possible to triple the number of conditions that led to a crystallization.

d. Statistics on the Crystals of the Different Proteins Studied on the HTXIab Platform

Table 8 below presents the number of conditions that led to crystals after conventional screening (576 commercial conditions for complex 10 and 480 for complex 31) at the HTXIab platform for the different proteins studied. The values indicated correspond to an observation of the crystallization plates after 85 days. The column “unique conditions” corresponds to the number of conditions that lead to crystallization in the presence of complexes, but no crystallization under the same conditions in the absence of complex (called natives).

TABLE 8 Protein Complex concentration Commercial concentration Total Native Protein (mg/ml) Organism protein Complex (mM) conditions hits * Lysozyme (HEWL) 20 Gallus yes 10 10 576 17 Proteinase K 20 T. Album yes 10 10 576 22 Pho Protease 1 11.5 P. Horikoshii no 10 10 576 82 Pho Protease 1 11.5 P. Horikoshii no 17 10 576 82 Glyoxylate hydroxyl 10 P. Furiosus no 10 10 576 38 pyruvate reductase Glyoxylate hydroxyl 10 P. Furiosus no 17 10 576 38 pyruvate reductase Thaumatin 20 T. Daniellii yes 10 10 576 4 Lysozyme (HEWL) 20 Gallus yes 31 10 480 9 Proteinase K 20 T. Album yes 31 10 480 16 * Condition leading to crystallization in the absence of complex (native conditions) ** Condition leading to crystallization with addition of complex

The conditions leading to crystallization correspond to conditions leading to the appearance of crystals potentially exploitable for diffraction experiments. Complex 10 thus allows a significant increase in the number of crystallization conditions for the following proteins: HEWL and Pho protease I. While its effect may appear to be less effective in the case of Proteinase K and Thaumatin, that is not the case. Indeed, the introduction of Complex 10 does not lead to a significant increase in the number of lanthanide hits, but the conditions obtained are largely different from native conditions (21 for Proteinase K and 2 for Thaumatin). We increase then the number of potential conditions for obtaining crystals of the protein of interest,

It should be noted that in the case of the Protease I protein with complex 10, gaining 113 conditions is covering a wide range of different crystallization conditions. This proves once again that complex 10 is compatible with all the physico-chemical conditions that can be found in commercial crystallization kits.

Complex 10, like complexes 17 and 31, have all a nucleating effect. However, complexes 17 and 31 are less effective than complex 10. For example, complex 31 appears to be less effective than complex 10 for proteinase K. However, the 4 conditions leading to crystallization in the presence of complex 31 are different from those leading to native crystals. Those crystals are potentially of better quality. Obtaining new conditions leading to crystallization is therefore a step forward.

D) Hand-Held Laboratory Crystallizations

Commercial proteins (Paragraph A.a.) were manually crystallized according to known customary crystallization conditions (Table 2). The crystallization conditions described in Table 2 were therefore reproduced in the presence of 10 or 17 complexes at a concentration of 10 mM. To achieve these manual crystallization ranges, the crystallization drops were prepared according to the following scheme: 1.5 μl protein solution +1.5 μl complex solution at a concentration of 10 mM+1.5 μl precipitant solution. In order to highlight a potential effect of lanthanide complexes on crystallization, the precipitating agent range has been adjusted to be at the edge of the crystallization zone and eventually extended beyond when an effect was observed.

Thus, in the case of the lysozyme protein, a marked nucleating effect was observed. The nucleating effect should be understood here as the growth of crystals in the presence of the lanthanide complex at low concentrations of precipitating agents, concentrations which do not allow the formation of native crystals. In order to clearly highlight this nucleation phenomenon induced by complexes according to the invention, phase diagrams have been made by determining the ranges of concentration of precipitating agents and of protein that allow the crystals to be obtained.

a. Phase Diagrams for Native Chicken's Egg White Lysozvme and in the Presence of 10 mM Complex 10 or Complex 17

Phase diagrams of the lysozyme protein obtained after 2 days and 15 days of growth are shown in FIG. 5:

Each crystallization condition was made in triplicate. After only two days of crystal growth, a clear difference was observed. In the presence of complex 10 or 17, crystals were obtained at both low concentrations of protein (5 mg/ml) and precipitating agent.

After 20 days of crystalline growth in the presence of complex 10 or 17 at 10 mM, crystals were obtained over the entire range evaluated. In particular, crystals have appeared for 5 mg/ml of lysozyme and for a precipitating concentration of 500 mM. By comparison, the absence of lanthanide complex only allows the formation of crystals up to 500 mM of NaCl and for concentrations of 20 mg/ml of protein.

For comparison purposes:

MRI complexes, which do not induce nucleating effects, are typically used at concentrations in the range of 50-300 mM.

-   -   Lanthanide tris-dipicolinate produced a new crystalline form of         lysozyme only when used at concentrations above 50 mM.     -   The MIPs proposed by Naomi E. Chayen (Saridakis, E., Khurshid,         S., Govada, L., Phan, Q., Hawkins, D., Crichlow, G. V., Lolis,         E., Reddy, S. M., & Chayen, N. E. (2011). Proceedings of the         National Academy of Sciences. 108,11081-11086) lead to         crystallization zone offsets of only one to two precipitating         agent concentration units. The associated nucleating effects are         obtained only for conventional concentrations of lysozyme in the         order of 30 mg/ml (Khurshid et al., Automating the application         of smart materials for protein crystallization, (2014) Acta         cryst D). According to Saridakis et al. (Saridakis, E.,         Khurshid, S., Govada, L, Phan, Q., Hawkins, D., Crichlow, G. V.,         Lolis, E., Reddy, S. M., & Chayen, N. E. (2011). Proceedings of         the National Academy of Sciences. 108,11081-11086.), the range         of crystallization of lysozyme in the presence of their         nucleating agents starts from 480 mM of NaCl at a protein         concentration of 20 mg/mL. No lysozyme crystal was observed         below 460 mM of NaCl. Concerning the thaumatin protein (30         mg/ml) no crystal is observed below 0.2 M tartrate. In the         presence of their nucleating agents, they obtain crystals for         0.3 and 0.4 M tartrate (idem complex 10) and native crystals for         0.5 M tartrate.     -   POM complexes have a nucleating effect only with high         concentrations of lysozyme (about 100 mg/ml), which poses         solubility problems for most proteins. (A. Bijelic et al         Chicken's Egg-White Lysozyme Crystallisation: Protein Stacking         and Structure Stability Enhanced by a Tellurium (VI)-Centred         Polyoxotungstate Chem Bio Chem 2015, 16,233-241).

b. Phase Diagram for Native Chicken's Egg White Lysozyme and in the Presence of 10 mM Complex 11 (Eu)

The phase diagram obtained in the presence of complex 11 (FIG. 6) is equivalent to that obtained in the presence of complex 10. The nature of lanthanide present within the complex does not influence the observed nucleating effect.

c. Phase Diagrams for Protein of Unknown Structure pb6

The protein pb6 has been purified according to the protocol described above. The condition pb6-1 (Paragraph Cb) obtained at the HTXIab robot was reproduced manually. Phase diagrams were determined. They are shown in FIG. 7. As with lysozyme, the nucleating effect is very important. The crystals obtained in the presence of the lanthanide complex according to the invention are very promising (see FIG. 8) for a crystallographic study.

Crystals obtained in the presence of 10 mM complex 10 are perfectly exploitable for diffraction experiments. Native crystals are too small, too thin and poorly organized. Thus, in the case of the protein pb6, a nucleating and crystallizing effect is observed induced by compound 10.

d. Crystallizing Effect of Lanthanide Complexes 10

If an improvement of crystallization (number of crystals, crystal size, diffraction improvement) is observed with the addition of lanthanide complexes, this is referred to a crystallization effect.

In the case of the crystallization of the Protease 1 protein of P. horikoshil, a crystallizing effect was also observed linked to the addition of complex 10. Indeed, the crystals obtained in the presence of 10 mM of complex 10 appeared on average 2.5 times larger than the crystals obtained under the same conditions, but without complex (average size evaluated on 10 crystals present in a photographed drop).

This crystallizing effect is therefore of great interest for X-ray crystallography since the diffraction intensity is proportional to the volume of the irradiated sample. This can thus provide a higher resolution for diffraction data.

E) Luminescence Properties and Applications.

The coordination complexes of terbium and europium (III) are known to have very particular luminescence properties, due to the f-f transitions that result in fine and characteristic emission lines of each element and long lifetimes (ps-ms). It is difficult to induce this luminescence by direct irradiation of the metal ion, because these f-f transitions are prohibited and therefore have very low molecular absorption coefficients. On the other hand, it is possible to sensitize this luminescence by an indirect process, called antenna effect, which consists in exciting an organic ligand containing a chromophore (typically an aromatic group) and transferring this energy to the metal ion (Luminescence of Lanthanide Ions in Coordination Compounds and Nanomaterials, Ed. A. De Bettencourt-Dias, Wiley 2014).

These luminescence properties of the proposed lanthanide complexes can be used in two steps in determining the structure of a protein: a) crystal detection during crystallization and b) crystal centering during the diffraction experiment.

a. Crystal Detection During Crystallization

Detection of crystals can sometimes be complicated, for example when the crystals are small, drowned into a precipitate or obtained at the edge of a crystallization drop. In the particular case of membrane proteins, we can also mention the problem of detecting crystals obtained by the crystallization technique of lipidic cubic phase.

To improve the crystals detection, many suppliers offer microscopes with a UV source, allowing the intrinsic fluorescence of aromatic amino acids, especially tryptophan. Examples include the UV source proposed by Molecular Dimension (http://www.moleculardimensions.com/applications/upload/Xtalight“100.pdf) or the UVEX microscope (http://www.moleculardimensions.com/shopdisplayproducts.asp? id=299&cat=UVEX+UV+Fluorescence+Imaging+systems) offered by the same company.

The use of lanthanide complexes luminescence can help to solve many of the problems mentioned above.

The luminescence of lanthanide complexes according to the invention has therefore been studied, using a UV source currently being marketed by NatXray on a conventional microscope and an external OceanOptics LED UV source (FIG. 9; Excitation wavelength 365 nm; Model LLS-365; http://oceanoptics.com/product/lls-family/) (lysozyme crystals obtained in the presence of 10 mM of complex 10 or MDH ANC80 protein crystals obtained in the presence of 50 mM of complex 17).

Using the NatX-ray system, the crystals obtained in the absence of Complex 10 appear blue (on the left). Conversely, those obtained with this complex appear green (on the right) which results in a contrast increase between the crystals and the surrounding solution. This can also be observed with the system using a UV LED source, since the crystals obtained in the presence of Complex 17 and under UV illumination are easier to identify than when observed in white light. For example, a small crystal is indicated by a white arrow (FIG. 9).

It should also be noted that luminescence is observable at two excitation wavelengths (280 nm and 365 nm).

b. Crystal Centering Aid

This part was evaluated using the IBS-ESRF CRYOBENCH instrument (http://www.isbg.fr/analyses-structurales/cryobench/) and the ESRF FIP-BM30A light line.

The crystals used (lysozyme crystals obtained in the presence of 10 mM of complex 10 or crystals of the protein MDH ANC80 obtained in the presence of 50 mM of complex 17) were conventionally frozen at 100 K on nylon loops.

To take pictures, the same external UV LED light source from OceanOptics was used. The quality of the photos obtained makes it possible to consider different ways for facilitating centering:

-   -   a direct centering through luminescence,     -   the use of a spectrophotometer to accurately measure         luminescence and search for areas with the highest intensities         corresponding to the presence of a crystal. The idea would be to         scan the loop with a rather thin UV beam.     -   Pre-positioning using luminescence, supplemented by precise         centering using the so-called Raster Scanning technique (Aishima         et al, Acta D (2010), D66,1032-1035), especially in the case of         small crystals or fine needle type crystals.

F) Phasing Potential of Lanthanide Complexes According to the Invention

a. Methodology Used

The evaluation of the phasing potential of lanthanide complexes according to the invention was carried out in a conventional manner, using different de novo phase-determining methods, representing a panel of commonly used techniques for the determination of biological macromolecular structures. This shows that the use of complexes according to the invention allows a habitual use of phasing methods.

The tested methods are:

-   -   The SAD method, which has the advantage of requiring a single         crystal and performing a single diffraction recording,     -   The MAD method, which has the advantage of requiring a single         crystal, which presupposes recording at several wavelengths and         which theoretically produces phases of better quality than the         SAD method,     -   The SIRAS method, which involves recording on a protein crystal         in the absence of complex and recording on a protein crystal in         the presence of complex. This method assumes an excellent         isomorphism of both crystals and therefore requires that they         have the same crystalline form.

Diffraction data were integrated and scaled with the XDS, SCALA and TRUNCATE programs.

The AutoSharp program (https://www.globalphasing.com/sharp/) has been used. This program automatically searches for the position of heavy atoms, refines them, determines the initial phases and improves them. The program was used with the default settings. The result of the phasing is evaluated on the basis of the merit figures (FOM for “Figure of Merit”), before and after phase improvement.

In a second step, the quality of the phasing was evaluated by automatically reconstructing the model of the protein under consideration. The number of residues modelled is then available compared to the expected number of residues. The Buccaneer program (http://www.ccp4.ac.uk/dist/html/cbuccaneer.html) was used with the default settings and with 10 rebuild cycles.

The diffraction data are conventionally recorded on a synchrotron light line. In order to determine the precise absorption threshold of lanthanide LIII of the lanthanide used, a fluorescence measurement was performed and processed using the Chooch program (http://www.gwyndafevans.co.uk/chooch.html). The recording wavelengths are thus obtained, in order to optimize the use of the lanthanide anomalous signal (SAD and MAD methods). In the case of the SIRAS method and in addition to recording at the LIII threshold of the lanthanide on the derivative crystal, a native crystal recording was made at the wavelength of 0.9798 Å. The results are shown in FIG. 10.

For each of the phasing methods evaluated, the recordings were made at the wavelengths indicated in Table 9:

TABLE 9 Phasing methods used and associated recording energies. Phasing methods Recordings made at energies corresponding to: SAD pk MAD pk, inf, rm SIRAS pk + native at 0.9798 Å

b. Crystallized Protein Diffraction Test in the Presence of 10 mM of Complex 10 or 17

Crystals of co-crystallized proteins in the presence of 10 mM of complex 10 or 17 (nucleating effect conditions of the lanthanide complex) were evaluated in terms of diffraction. The results of this assessment are summarized in Table 10:

TABLE 10 Results of diffraction tests obtained on different crystallized proteins in the presence of lanthanide complexes Protein Resolution (Å) HEWL 10m Complex 10 >1.5 Thaumatin 10 mM Complex 10 >1.5 Proteinase K 10 mM Complex 10 >1.5 Protease1 10 mM Complee 10 1.7 PfuGR 10 mM Complex 10 2.0 pb6-1 10 mM Complex 10 2.6 ANC80-1 10 mM Complex 17 1.7

In the case of Protease 1 protein, the crystallizing effect manifests itself both by an increase in the average crystal size, as indicated above, and by the resolution obtained for diffraction. The diffraction data recorded on a crystal obtained in the presence of 10 mM of complex 10 has a resolution of 1.7 Å. The best model currently available in the Protein Data Bank is at 2.0 Å (PDB code: 1G2I Publication reference: idem protocol part 1b).

c. De Novo Phasing of Model Proteins

The structures of the different model proteins were determined according to the different methods explained in paragraph F.a.

In addition, phasing attempts were carried out on crystals obtained under nucleating conditions of the complex (i.e. at 10 mM), but the possibility of soaking a crystal obtained in the presence of 10 mM of complex 10 or 17 in a solution similar to the crystallization condition and containing 100 mM of the lanthanide complex was also studied. This was intended to eventually increase the protein's tagging rate and thus facilitating the determination of its structure. The soaking time was about one minute. It should be noted that this soaking technique can also be applied to native crystals (obtained in the absence of lanthanide complex).

To summarize, the following three crystal preparation methods were evaluated for a de novo phasing using lanthanide complexes according to the invention:

-   -   co-crystallized crystal in the presence of 10 mM of complex         (nucleating condition),     -   co-crystallized crystal in the presence of 10 mM of complex and         soaked in a solution containing 100 mM of the same complex,     -   native crystal soaked in a 100 mM solution of the lanthanide         complex.

The diffraction data set was obtained in accordance with the methodology explained in paragraph F. a. The results are presented in Table 11 below.

TABLE 11 Results of de novo phasing for model proteins FOM Buccanneer FOM after % of Resolution after SOLOM rebuilt Protein Complex Concentration Method (Å) SHARP ON model Hewl 10 10 mM SAD 1.8 0.428 0.881 79.1 Hewl 17 10 mM SAD 1.8 0.314 0.839 46.5 Protease1 17 Soaking * SAD 2.0 0.389 0.955 95 PfuGR 17 Soaking* of SIRAS 2.5 0.043 0.943 68 native crystal PfuGR 10 Soaking * SAD 2.5 0.184 0.923 84.5 * Soaking: In a cryoprotectant solution containing 100 mM of complex

The high phasing power of the complex objects of the present invention is reflected in the percentage of model reconstructed without manual intervention. The more the quality of the phase determination (depending on the phasing method used, the occupancy of the complex fixing sites, the data quality, etc.), the more the experimental electronic density map can be interpreted by automatic reconstruction programs (such as the Buccaneer program). In the evaluated cases, models automatically rebuilt from 50% to almost 100% of the final model have been obtained.

d. Examples of Electronic Density Obtained After Phasing and Solvent Flattening

An example of experimental electron density obtained using the methods described above for glyoxylate reductase protein and Protease 1 protein are shown in FIG. 11. The glyoxylate reductase crystals were obtained under the conditions described in Table 4 in the presence of 10 mM of complex 10. The Protease 1 protein crystals were obtained under the conditions described in Table 4 in the presence of 10 mM of complex 10 and then soaked in a cryoprotective solution containing 100 mM of complex 10.

In both cases, given the quality of phase determination, easily interpretable experimental electronic density maps are obtained, where one can distinguish side chains of amino acids. A tyrosine for the glyoxylate reductase map and a tryptophan for the Protease 1 Protein map. The images were produced using the coot software.

G) Application of the Technology to the Determination of the Structure of the MDH ANC80 Protein

a. Nucleating and Crystallizing Effect: the Case of MDH ANC80

The MDH ANC80 protein concentrated at 10 mg/ml was sent to the crystallization robot for a conventional screening of the 576 conditions. The most promising crystallization condition is the condition ANC-1 in Table 7. The photos obtained in the crystallization robot for this condition are shown in FIG. 12.

This condition was reproduced manually in the laboratory. Crystals appeared in the presence of 10 mM of complex 17 and 10 mM of complex 10 in 7 days. The same condition in native condition (without lanthanide complex) was also performed. After about 3 weeks, crystals of different shapes appeared (not shown).

The native crystals have been tested for diffraction. The resolution is in the order of 2.5 Å. Those crystals have a different symmetry from that obtained for crystals obtained in the presence of complex 10 or 17 (Space group F222) with mesh parameters of 81,140 and 395 Å respectively for a, b, and c. This is compared to the space group obtained for crystals in the presence of complex and the resolution obtained for diffraction data. The crystals in the presence of 10 mM of lanthanide complex diffract at 1.7 Angstrom. Thus, the nucleating and crystallizing effects are properly observed in the case of this protein.

The structure of the ANC80 MDH was determined using the MAD method. Three datasets were recorded on the same crystal at the terbium absorption threshold LIII. An additional dataset was measured at the selenium absorption threshold K to obtain the best possible resolution. The statistics, after integration across all the data collected, are presented in Table 12 below.

TABLE 12 Data collection, phasing and refinement statistics ANC80 ANC80 10 mM complex 17 10 mM complex 17 Data collection Se threshold K Tb threshold Line id23-2 (ESRF) L_(III). Line BM30A (ESRF) Space group R3 R3 Mesh parameters a, b, c (Å) a = b = 217.5 a = b = 217.5 c = 86.3 c = 86.3 pk* inf rm Wavelength 0.8726 1.650237 1.65087 1.64766 Resolution (Å) 1.85 2 2 2 R_(merge) 0.093 (0.850) 0.126 (0.617) 0.117 (1.035) 0.116 (1.111) I/sigma(I) 7.3 (1.3) 6.8 (2.1)   7 (1.5) 7.6 (1.3) Completeness (%) 98.9 (97.6) 99.5 (96.8) 99.6 (97.0) 99.5 (96.5) Multiplicity 3.9 (3.9) 5.5 (5.2) 5.5 (5.1) 5.5 (5.0) Automatic rebuilt 99.2% of the model rebuilt at 2.0 Angström (Buccaneer) *according to FIG. 8

The refinement of the model leads to very good quality factors with a R and Rfree factor of 19.2% and 21.2%.

The following luminescent complexes have also been tested at 10 mM and do not have any effect on crystallization:

ANNEXE 1 Amino acid sequence of the different proteins tested Lysozyme (Gallus gallus) HEWL: SEQ ID No 1 KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSR WWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQ AWIRGCRL Thaumatine (T. danielli): SEQ ID No 2 ATFEIVNRCSYTVWAAASKGDAALDAGGRQLNSGESWTINVEPGTNGGKIWARTDCYFDDS GSGICKTGDCGGLLRCKRFGRPPTTLAEFSLNQYGKDYIDISNIKGFNVPMNFSPTTRGCRGVR CAADIVGQCPAKLKAPGGGCNDACTVFQTSEYCCTTGKCGPTEYSRFFKRLCPDAFSYVLDKP TTVTCPGSSNYRVTFCPTA Proteinase K (Tritirachium Album): SEQ ID No 3 AAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASHPEFEGRAQMVKTYYYSS RDGNGHGTHCAGTVGSRTYGVAKKTQLFGVKVLDDNGSGQYSTIIAGMDFVASDKNNRNCP KGWASLSLGGGYSSSVNSAAARLQSSGVMVAVAAGNNNADARNYSPASEPSVCTVGASDRY DRRSSFSNYGSVLDIFGPGTDILSTWIGGSTRSISGTSMATPHVAGLAAYLMTLGKTTAASACR YIADTANKGDLSNIPFGTVNLLAYNNYQA Glyoxylate and Hydroxypyruvate Reductase (P. furiosus): SEQ ID No 4 MKPKVFITRAIPENGINMLEEEFEVEVWEEEREIPREKLLEKVKDVDALVTMLSERIDQEVFENA PRLRIVANYAVGYDNIDVEEATRRGIYVTNTPDVLTNATADHAFALLLATARHVVKGDKFVRS GEWKRKGIAWHPKWFLGYELYGKTIGIVGFGRIGQAIARRAKGFNMRILYYSRTRKSQAEKEL GAEYRPLEEVLKESDFVILAVPLTKEIMYMINEERLKLMKPTAILVNIARGKVVDTKALIKALKE GWIAGAGLDVFEEEPYYNEELFSLDNVVLTPHIGSATFEAREAMAELVARNLIAFKRGEIPPTLV NKEVIKIRKPGFNEQ Protease 1 (P. horikoshii OT3): SEQ ID No 5 MKVLFLTANEFEDVELIYPYHRLKEEGHEVYIASFERGTITGKHGYSVKVDLTFDKVNPEEFDAL VLPGGRAPERVRLNEKAVSIARKMFSEGKPVASICHGPQILISAGVLRGRKGTSYPGIKDDMIN AGVEWVDAEVVVDGNWVSSRVPADLYAWMREFVKLLK Pb6 major protein of the phage T5 tail : SEQ ID No 6 MSLQLLRNTRIFVSTVKTGHNKTNTQEILVQDDISWGQDSNSTDITVNEAGPRPTRGSKRFN DSLNAAEWSFSTYILPYKDKNTSKQIVPDYMLWHALSSGRAINLEGTTGAHNNATNFMVNFK DNSYHELAMLHIYILTDKTWSYIDSCQINQAEVNVDIEDIGRVTWSGNGNQLIPLDEQPFDPD QIGIDDETYMTIQGSYIKNKLTILKIKDMDTNKSYDIPITGGTFTINNNITYLTPNVMSRVTIPIG SFTGAFELTGSLTAYLNDKSLGSMELYKDLIKTLKVVNRFEIALVLGGEYDDERPAAILVAKQAH VNIPTIETDDVLGTSVEFKAIPSDLDAGDEGYLGFSSKYTRTFINNLIVNGDGATDAVTAITVKS AGNVTTLNRSATLQMSVEVTPSSARNKEVTWAITAGDAATINATGLLRADASKTGAVTVEATA KDGSGVKGTKVITVTAGG ANC80 Malate Dehydrogenase (synthetic protein): SEQ ID No 7 MTKVSWGAAGIVGAAAGYNLALRDIADELVFVDIPDQEDVTIGQAADTNHGVAYDSNTIVR QGGYEDTAGSDVVVITAGIPRQPGQTRIDLAGDNAPIMEDIGSSLAEHNDDFVTITTSNPVDL LNRHLYETGDRAREKVIGFGGRLDSARFRYVLSQRFDAPVQNVEATILGEHGDAQVPVFSKVR VDGTDPEFSADEKEEILGDLQESAMDVIERKGATQWGPATGVAHMVEAVLHDTGEVLPGSVV LDGEFGHEDTAFGVPVKLGSNGVEEVVEWDLDDYEQDLMDDAAEKLSDQYDKIA

ANNEXE 2A Well C U Salt C U Buffer pH C U Precipitant C U Additive ANNEXE: ‘QUIAGEN” plate B09 0.2 M Ammonium 0.1 M TRIS 8.5 50 % (v/v) MPD phosphate B05 0.2 M Ammonium acetate 0.1 M tri-Sodium citrate 5.6 30 % (v/v) MPD B08 0.5 M Ammonium sulfate 0.1 M HEPES 7.5 30 % (v/v) MPD B06 0.2 M Magnesium acetate 0.1 M Sodium cocaylate 6.5 10 % (v/v) MPD G08 10 % (w/v) PEG 1000 10 % (w/v) PEG 8000 H11 0.1 M HEPES 7.5 20 % (w/v) PEG 8 % (v/v) Ethylene glycol G09 30 % (w/v) PEG 1500 G11 0.2 M Ammonium sulfate 0.1 M Sodium acetate 4.6 30 % (w/v) PEG 2000 MME G10 0.01 M Nickel chloride 0.1 M TRIS 8.5 20 % (w/v) PEG 2000 MME H12 0.1 M MES 6.5 12 % (w/v) PEG 20000 G02 0.2 M Calcium chloride 0.1 M HEPES sodium 7.5 28 % (v/v) PEG 400 salt G04 0.2 M Magnesium chloride 0.1 M HEPES sodium 7.5 30 % (v/v) PEG 400 salt G05 0.2 M tri-Sodium citrate 0.1 M TRIS HCL 8.5 30 % (v/v) PEG 400 G01 2 % (v/v) PEG 400 0.1 M HEPES sodium 7.5 2 M Ammonium salt sulfate G03 0.1 M Cadmium chloride 0.1 M Sodium actetate 4.6 30 % (w/v) PEG 400 H02 0.2 M Ammonium acetate 0.1 M Sodium acetate 4.6 30 % (v/v) PEG 4000 H01 0.2 M Ammonium sulfate 0.1 M Sodium actate 4.6 25 M PEG 4000 H05 0.2 M Lithium sulfate 0.1 M TRIS HCL 8.5 30 % (w/v) PEG 4000 H06 0.2 M Sodium acetate 0.1 M TRIS HCL 8.5 30 % (w/v) PEG 4000 H03 0.2 M Ammonium acetate 0.1 M tri-Sodium citrate 5.6 30 % (w/v) PEG 4000 H07 0.2 M Ammonium sulfate 30 % (w/v) PEG 4000 G12 0.1 M Sodium acetate 4.6 8 % (w/v) PEG 4000 H04 0.2 M Magnesium chloride 0.1 M TRIS HCL 8.5 30 % (w/v) PEG 4000 H08 0.2 M Ammonium sulfate 0.1 M MES 6.5 30 % (w/v) PEG 5000 MME G06 0.1 M Sodium chloride 0.1 M BICINE 9 20 M PEG 550 MME G07 0.01 M Zinc sulfate 0.1 M MES 6.5 25 M PEG 550 MME H09 0.1 M HEPES 7.5 10 % (v/v) PEG 6000 5 % (v/v) MPD H10 10 (w/v) PEG 6000 2 M Sodium chloride F04 0.1 M HEPES 7.5 10 % (w/v) PEG 8000 F06 0.2 M Zinc acetate 0.1 M MES 6.5 18 % (w/v) PEG 8000 F10 0.2 M Ammonium sulfate 0.1 M MES 6.5 30 % (w/v) PEG 8000 F07 0.2 M Calcium acetate 0.1 MES 6.5 18 % (w/v) PEG 8000 F08 0.2 M Magnesium acetate 0.1 M MES 6.5 20 % (w/v) PEG 8000 F11 0.2 M Sodium acetate 0.1 MES 6.5 30 % (w/v) PEG 8000 F12 0.2 M Ammonium sulfate 30 % (w/v) PEG 8000 F05 0.5 M Lithium sulfate 15 % (w/v) PEG 8000 F09 0.05 M Potassium 20 % (w/v) PEG 8000 phosphate F03 0.1 M Tris HCl 8.5 8 % (w/v) PEG 8000 D03 0.05 M Cadmium sulfate 0.1 M HEPES 7.5 1 M Sodium acetate D02 0.1 M Imidazole 6.5 1 M Sodium acetate D04 0.1 M MES 6.5 1.4 M Sodium acetate D07 0.1 M HEPES 7.5 4.3 M Sodium chloride D06 0.1 M Sodium phosphate 0.1 M MES 6.5 2 M Sodium chloride 0.1 M Sodium phosphate D05 0.1 M Sodium acetate 4.6 2 M Sodium chloride D11 0.1 M Sodium acetate 4.6 2 M Sodium formate D12 4 M Sodium formate D10 0.1 M HEPES sodium 7.5 0.8 % (w/v) Sodium 0.8 M Potassium salt phosphate phosphate B11 0.1 TRIS 8.5 25 % (v/v) tert-Butanol B12 0.1 M tri-Sodium citrate 5.6 35 % (v/v) tert-Butanol D08 0.1 M HEPES sodium 7.5 1.4 M tri-Sodium citrate salt D09 1.6 M tri-Sodium citrate pH6.5 A03 0.2 M Magnesium chloride 0.1 TRIS 8.5 3.4 M 1,6-Hexanediol A02 0.1 M Tri-Sodium 5.6 2.5 M 1,6-Hexanediol citrate A01 0.01 M Cobalt chloride 0.1 M Sodium acetate 4.6 1 M 1,6-Hexanediol C04 0.1 M HEPES 7.5 2 M Ammonium formate C03 0.1 TRIS HCL 8.5 2 M Ammonium phosphate C02 0.1 M Tri-Sodium 5.6 1 M Ammonium citrate phosphate C01 0.4 M Ammonium phosphate C08 0.1 M Sodium chloride 0.1 HEPES 7.5 1.6 M Ammonium sulfate C09 0.01 M Cobalt chloride 0.1 MES 6.5 1.8 M Ammonium sulfate C05 0.1 Sodium acetate 4.6 2 M Ammonium sulfate C06 0.1 M TRIS.HCL 8.5 2 M Ammonium sulfate C10 0.2 M K/Na tartrate 0.1 M Tri-Sodium 5.6 2 M Ammonium citrate sulfate C07 M 2 M Ammonium sulfate E06 0.5 M Sodium chloride, M 0.01 M CTAB 0.01 M Magnesium chloride E02 10 % (v/v) Dioxane, 0.1 M MES 6.5 1.6 M Ammonium sulfate E03 M 35 % (v/v) Dioxane E01 2 % (v/v) Dioxane, 0.1 M BICINE 9.0 10 % (w/v) PEG 20000 A12 10 % (v/v) ethanol M 1.5 M Sodium chloride B01 0.1 TRIS 8.5 20 % (v/v) Ethanol B02 M 25 % (v/v) Ethylene glycol E04 0.5 M Sodium chloride 0.1 tri-Sodium citrate 5.6 2 M Ethylene imine polymer E05 12 (v/v) Glycerol, 0.1 M TRIS 8.5 1.5 M Ammonium sulfate C11 M 7.0 1 M Imidazole A10 0.2 M Magnesium chloride 0.1 M HEPES sodium 7.5 30 % (v/v) Isopropanol salt A08 0.2 M tri-Sodium citrate 0.1 M HEPES sodium 7.5 20 % (v/v) Isopropanol salt A05 10 % (v/v) Isopropanol, 0.1 M HEPES sodium 7.5 20 % (w/v) PEG 9000 salt A06 0.2 M Calcium chloride 0.1 M Sodium acetate 4.6 20 % (v/v) Isopropanol A09 0.2 M tri-sodium citrate 0.1 M MES 6.5 30 % (v/v) Isopropanol A11 0.2 M ammonium acetate 0.1 M TRIS HCL 8.5 30 % (v/v) Isopropanol A07 20 % (v/v) isopropanol 0.1 M Tri-sodium 5.6 20 % (w/v) PEG 4000 citrate A04 5 % (v/v) isopropanol 2 M Ammonium sulfate E08 0.1 M HEPES 7.5 20 % (v/v) Jeffamine M-600 E07 0.01 M ferric chloride 0.1 M Tri_sodium 5.6 10 % (v/v) Jeffamine M-600 citrate D01 0.1 M HEPES sodium 7.5 0.8 M K/Na tartare salt C12 0.4 M K/Na tartare E11 0.1 M HEPES sodium 7.5 1.5 M Lithium sulfate salt E10 0.01 M nickel chloride 0.1 M TRIS 8.5 1 M Lithium sulfate E09 0.5 M 0.1 M Tri-sodium 5.6 1 M Lithium sulfate citrate E12 0.1 M BICINE 9 2 M Magnesium chloride F01 0.2 M Magnesium formate F02 0.1 M MES 6.5 1.6 M Magnesium sulfate B10 0.1 M HEPES 7.5 70 % (v/v) MPD B07 0.2 M tri-sodium citrate 0.1 M HEPES sodium 7.5 30 % (v/v) MPD salt B03 0.02 M clacium chloride 0.1 M Sodium acetate 4.6 30 % (v/v) MPD B04 0.2 M sodium chloride 0.1 M Sodium acetate 4.6 30 % (v/v) MPD ANNEXE: “Hampton 2” plate A01 0.02 M calcium chloride 0.1 M Sodium acetate 4.6 15 % (v/v) MPD dihydrate trihydrate A02 0.2 M ammonium acetate 0.1 M Tri-Sodium citrate 5.6 15 % (w/v) PEG 4000 dihydrate A03 0.2 M lithium sulfate 0.1 M TRIS HCL 8.5 15 % (w/v) PEG 4000 monohydrate A04 0.1 M Imidazole 6.5 0.5 M Sodium acetate trihydrate A05 2 M Sodium formate A06 5 % (v/v) iso-propanol 0.1 M HEPES sodium 7.5 10 % (w/v) PEG 4000 salt A07 0.2 M sodium fluoride 7.1 20 % (w/v) PEG 3350 A08 0.2 M ammonium chloride 6.3 20 % (w/v) PEG 3350 A09 0.2 M sodium nitrate 6.8 20 % (w/v) PEG 3350 A10 0.2 M magnesium acetate 7.7 20 % (w/v) PEG 3350 tetrahydrate A11 0.2 M sodium sulfate 6.6 20 % (w/v) PEG 3350 decahydrate A12 0.2 M potassium 4.7 20 % (w/v) PEG 3350 dihydrogen phosphate B01 0.2 M Potassium/ sodium tartrate tetrahydrate B02 0.2 M ammonium acetate 0.1 M Sodium acetate 4.6 15 % (w/v) PEG 4000 trihydrate B03 0.2 M magnesium acetate 0.1 M Sodium cacodylate 6.5 10 % (w/v) PEG 8000 tetrahydrate B04 0.2 M ammonium acetate 0.1 M Tri-Sodium citrate 5.6 15 % (w/v) MPD dihydrate B05 0.1 M Sodium acetate 4.6 1 M sodium formate trihydrate B06 0.05 M potassium 10 % (w/v) PEG 8000 dihydrogen phosphate B07 0.2 M potassium fluoride 7.2 20 % (w/v) PEG 3350 B08 0.2 M sodium iodide 6.9 20 % (w/v) PEG 3350 B09 0.2 M potassium nitrate 6.9 20 % (w/v) PEG 3350 B10 0.2 M zinc acetate 6.3 20 % (w/v) PEG 3350 dihydrate B11 0.2 M potassium sulfate 6.7 20 % (w/v) PEG 3350 B12 0.2 M di-potassium 9.2 20 % (w/v) PEG 3350 hydrogen phosphate C01 0.2 M Ammonium dihydrogen phosphate C02 0.1 M Tri-Sodium citrate 5.6 0.5 M Ammonium dihydrate dihydrogen phosphate C03 0.2 M ammonium acetate 0.1 M TRIS HCL 8.5 15 % (v/v) Iso-propanol C04 0.2 M tri-Sodium citrate 0.1 M HEPES sodium 7.5 10 % (v/v) iso-propanol dihydrate salt C05 0.1 M HEPES sodium 7.5 0.4 M sodium 0.4 M Potassium salt dihydrogen dihydrogen phosphate phosphate C06 15 % (w/v) PEG 1500 C07 0.2 M ammonium fluoride 6.2 20 % (w/v) PEG 3350 C08 0.2 M potassium iodide 6.8 20 % (w/v) PEG 3350 C09 0.2 M ammonium nitrate 6.3 20 % (w/v) PEG 3350 C10 0.2 M sodium acetate 7.9 20 % (w/v) PEG 3350 trihydrate C11 0.2 M ammonium sulfate 6 20 % (w/v) PEG 3350 C12 0.2 M ammonium 4.6 20 % (w/v) PEG 3350 dihydrogen phosphate D01 0.1 M TRIS HCL 8.5 1 M ammonium sulfate D02 0.2 M magnesium chloride 0.1 M HEPES sodium 7.5 15 % (v/v) iso-propanol hexahydrate salt D03 0.2 M ammonium sulfate 0.1 M Sodium acetate 4.6 12.5 % (w/v) PEG 4000 trihydrate D04 0.2 M sodium acetate 0.1 M Sodium 6.5 15 % (w/v) PEG 8000 trihydrate cacodylate D05 0.1 M TRIS HCL 8.5 4 % (w/v) PEG 8000 D06 0.1 M magnesium formate D07 0.2 M lithium chloride 6.7 20 % (w/v) PEG 3350 anhydrous D08 0.2 M ammonium iodide 6.2 20 % (w/v) PEG 3350 D09 0.2 M magnesium formate 5.9 20 % (w/v) PEG 3350 D10 0.2 M calcium acetate 7.3 20 % (w/v) PEG 3350 hydrate D11 0.2 M di-sodium tartate 7.2 20 % (w/v) PEG 3350 dihydrate D12 0.2 M di-ammonium 7.9 20 % (w/v) PEG 3350 hydrogen phosphate E01 0.2 M tri-sodium citrate 0.1 M HEPES sodium 7.5 15 % (v/v) MPD dihydrate salt E02 0.2 M tri-sodium citrate 0.1 M TRIS HCl 8 15 % (v/y) PEG 400 dihydrate E03 0.2 M magnesium acetate 0.1 M sodium 6.5 15 % (v/v) MPD tetrahydrate cacodylate E04 0.1 M HEPES sodium 7.5 0.4 M potassium/ salt sodium tartrate tetrahydrate E05 0.1 M sodium acetate 4.6 4 % (w/v) PEG 4000 trihydrate E06 0.2 M zinc acetate 0.1 M sodium 6.5 9 % (w/v) PEG 8000 dihydrate cacodylate E07 0.2 M magnesium chloride 5.8 20 % (w/v) PEG 3350 hexahydrate E08 0.2 M sodium thiocyanate 6.9 20 % (w/v) PEG 3350 E09 0.2 M sodium formate 7.2 20 % (w/v) PEG 3350 E10 0.2 M potassium acetate 7.8 20 % (w/v) PEG 3350 E11 0.2 M potassium sodium 7.2 20 % (w/v) PEG 3350 tartrate tetrahydrate E12 0.2 M tri-lithium citrate 8.1 20 % (w/v) PEG 3350 tetrahydrate F01 0.2 M magnesium chloride 0.1 M TRIS HCl 8.5 15 % (w/v) PEG 4000 hexahydrate F02 0.2 M calcium chloride 0.1 M HEPES sodium 7.5 14 % (v/v) PEG 400 dihydrate salt F03 0.2 M sodium acetate 0.1 M TRIS HCl 8.5 15 % (w/v) PEG 4000 trihydrate F04 0.2 M ammonium sulfate 15 % (w/v) PEG 8000 F05 0.1 M HEPES sodium 7.5 0.7 M tri-sodium citrate salt dihydrate F06 0.2 M calcium acetate 0.1 M Sodium 6.5 9 % (w/v) PEG 8000 hydrate cacodylate F07 0.2 M sodium chloride 6.9 20 % (w/v) PEG 3350 F08 0.2 M potassium 7 20 % (w/v) PEG 3350 thiocyanate F09 0.2 M potassium formate 7.3 20 % (w/v) PEG 3350 F10 0.2 M ammonium acetate 7.1 20 % (w/v) PEG 3350 F11 0.2 M di-ammonium 6.6 20 % (w/v) PEG 3350 tartrate F12 0.2 M tri-sodium citrate 8.2 20 % (w/v) PEG 3350 dihydrate G01 0.1 M Sodium 6.5 0.7 M sodium acetate cacodylate trihydrate G02 0.2 M ammonium sulfate 0.1 N Sodium 6.5 15 % (w/v) PEG 8000 cacodylate G03 0.2 M magnesium chloride 0.1 M HEPES sodium 7.5 15 % (w/v) PEG 400 hexahydrate salt G04 0.2 M ammonium sulfate 15 % (w/v) PEG 4000 G05 2 % (v/v) PEG 400 0.1 M HEPES sodium 7.5 1 M Ammonium salt sulfate G06 0.1 M Sodium acetate 4.6 1 M Ammonium trihydrate sulfate G07 0.2 M Calcium chloride 5.1 20 % (w/v) PEG 3350 dihydrate G08 0.2 M Lithium nitrate 7.1 20 % (w/v) PEG 3350 G09 0.2 M Ammonium formate 6.6 20 % (w/v) PEG 3350 G10 0.2 M Lithium sulfate 5.4 20 % (w/v) PEG 3350 monohydrate G11 0.2 M Sodium dihydrogen 4.5 20 % (w/v) PEG 3350 phosphate monohydrate G12 0.2 M Tri-potassium citrate 8.3 20 % (w/v) PEG 3350 monohydrate H01 0.2 M Tri-sodium citrate 0.1 M Sodium 6.5 15 % (v/v) iso-propanol dihydrate cacodylate H02 0.1 M HEPES sodium 7.5 0.75 M lithium sulfate salt monohydrate H03 0.2 M Calcium chloride 0.1 M Sodium acetate 4.6 10 % (v/v) iso-propanol dihydrate trihydrate H04 1 M ammonium sulfate H05 10 % (v/v) Iso-propanol 0.1 M Tri-sodium 5.6 10 % (w/v) PEG 4000 citrate dihydrate H06 0.1 M TRIS HCl 8.5 1 M Ammonium dihydrogen phosphate H07 0.2 M Potassium chloride 6.9 20 % (w/v) PEG 3350 H08 0.2 M Magnesium nitrate 5.8 20 % (w/v) PEG 3350 hexahydrate H09 0.2 M Lithium acetate 7.8 20 % (w/v) PEG 3350 dihydrate H10 0.2 M Magnesium sulfate 5.9 20 % (w/v) PEG 3350 heptahydrate H11 0.2 M Di-sodium hydrogen 9.1 20 % (w/v) PEG 3350 phosphate dihydrate H12 0.2 M Di-ammonium 5 20 % (w/v) PEG 3350 hydrogen citrate ANNEXE: ‘Hampton 3” plate A01 0.1 M Sodium chloride 0.1 M Sodium acetate 4.6 12 % (v/v) MPD trihydrate A02 0.1 M Lithium sulfate 0.1 M Sodium acetate 4.6 1 M ammonium monohydrate trihydrate dihydrogen phosphate A03 0.1 M Sodium chloride 0.1 M Tri-sodium 5.6 12 % (w/v) PEG 4000 citrate dihydrate A04 0.1 M Magnesium chloride 0.1 M ADA 6.5 12 % (w/v) PEG 6000 hexahydrate A05 0.1 M Ammonium sulfate 0.1 M HEPES sodium 7.5 18 % (v/v) PEG 400 salt A06 0.1 M Di-ammonium 0.1 M TRIS HCl 8.5 0.5 M di-sodium 0.5 M Di-potassium hydrogen hydrogen hydrogen phosphate phosphate phosphate A07 0.01 M Magnesium chloride 0.05 M MES 5.6 2 M lithium sulfate hexahydrate monohydrate A08 0.1 M Potassium chloride 0.05 M MES 6 10 % (v/v) PEG 400 0.01 M Magnesium chloride hexahydrate A09 0.2 M Potassium chloride 0.05 M Sodium 6 1 % (w/v) PEG 4000 0.01 M Calcium chloride cacodylate dihydrate A10 0.08 M Magnesium acetate 0.05 M Sodium 6.5 30 % (w/v) PEG 4000 0 0 tetrahydrate cacodylate A11 0.2 M Ammonium chloride 0.05 M HEPES sodium 7 30% % (w/v) 1,6-hexanediol 0.01 M Magnesium chloride salt hexahydrate A12 0.1 M Potassium chloride 0.05 M TRIS HCl 7.5 10 % (v/v) PEG monomethyl 0.015 M Magnesium chloride ether 550 hexahydrate B01 0.1 M Zinc acetate 0.1 M Sodium acetate 4.6 12 % (w/v) PEG 4000 dihydrate trihydrate B02 0.1 M Sodium chloride 0.1 M Sodium acetate 4.6 12 % (w/v) PEG 6000 trihydrate B03 0.1 M Lithium sulfate 0.1 M Tri-sodium 5.6 12 % (w/v) PEG 6000 monohydrate citrate dihydrate B04 0.1 M ADA 6.5 12 % (v/v) MPD B05 0.1 M Ammonium sulfate 0.1 M HEPES sodium 7.5 10 % (w/v) PEG 4000 salt B06 0.1 M TRIS HCl 8.5 0.1 M sodium acetate trihydrate B07 0.01 M Magnesium acetate 0.05 M MES 5.6 2.5 M ammonium tetrahydrate sulfate B08 0.005 M Magnesium sulfate 0.05 M MES 6 5 % (w/v) PEG 4000 0 B09 0.01 M Magnesium acetate 0.05 M Sodium 6.5 1.3 M lithium sulfate 0 0 tetrahydrate cacodylate monohydrate B10 0.2 M Potassium chloride 0.05 M Sodium 6.5 10 % (w/v) PEG 8000 0.1 M Magnesium acetate cacodylate tetrahydrate B11 0.1 M Potassium chloride 0.05 M HEPES sodium 7 15 % (v/v) MPD 0.005 M Magnesium sulfate aq. salt B12 0.01 M Magnesium acetate 0.05 M TRIS HCl 7.5 5 % (v/v) iso-propanol 0 0 tetrahydrate C01 0.2 M Ammonium sulfate 0.1 M Sodium acetate 4.6 10 % (w/v) PEG 4000 trihydrate C02 0.1 M Magnesium chloride 0.1 M Sodium acetate 4.6 12 % (w/v) PEG 600 hexahydrate trihydrate C03 0.1 M Magnesium chloride 0.1 M Tri-sodium 5.6 4 % (v/v) MPD hexahydrate citrate dihydrate C04 0.1 M Lithium sulfate 0.1 M ADA 6.5 1 M magnesium monohydrate sulfate hydrate C05 0.1 M tri-sodium citrate 0.1 M HEPES sodium 7.5 12 % (v/v) MPD dihydrate salt C06 0 0 0.1 M TRIS HCl 8.5 0.1 M sodium chloride C07 0.1 M magnesium acetate 0.05 M MES 5.6 20 % (v/v) MPD tetrahydrate C08 0.01 M magnesium chloride 0.05 M sodium 6 1 M lithium sulfate 0 0 hexahydrate cacodylate monohydrate C19 0.01 M magnesium sulfate 0.05 M sodium 6.5 2 M ammonium 0 0 cacodylate sulfate C10 0.2 M ammonium acetate 0.05 M sodium 6.5 30 % (w/v) PEG 8000 0.01 M magnesium acetate cacodylate tetrahydrate C11 0.1 M potassium chloride 0.05 M HEPES sodium 7 5 % (v/v) PEG 400 0.01 M magnesium chloride salt hexahydrate C12 0.05 M ammonium acetate 0.05 M TRIS HCl 7.5 10 % (v/v) MPD 0.01 M magnesium chloride hexahydrate D01 0.1 M sodium chloride 0.1 M sodium acetate 4.6 12 % (v/v) iso-propanol trihydrate D02 0.1 M sodium chloride 0.1 M tri-sodium citrate 5.6 18 % (v/v) PEG 400 dihydrate D03 0 0 0.1 M tri-sodium citrate 5.6 0.1 M sodium chloride dihydrate D04 0.3 M lithium sulfate 0.1 M ADA 6.5 4 % (v/v) PEG 400 monohydrate D05 0 0 0.1 M HEPES sodium 7.5 1 M tri-sodium citrate salt dihydrate D06 0.1 M di-ammonium 0.1 M TRIS HCl 8.5 12 % (w/v) PEG 6000 hydrogen phosphate D07 0.2 M potassium chloride 0.05 M MES 5.6 10 % (v/v) PEG 400 0.01 M magnesium sulfate D08 0.01 M magnesium sulfate 0.05 M sodium 6 1.8 M lithium sulfate 0 0 cacodylate monohydrate D09 0.1 M ammonium acetate 0.05 M sodium 6.5 10 % (v/v) iso-propanol 0.015 M magnesium acetate cacodylate tetrahydrate D10 0.05 M Magnesium sulfate 0.05 M HEPES sodium 7 1.6 M Lithium sulfate 0 0 aq. salt monohydrate D11 0.1 M Potassium chloride 0.05 M HEPES sodium 7 10 % (v/v) PEG 400 0.01 M Calcium chloride salt dihydrate D12 0.2 M Potassium chloride 0.05 M TRIS HCl 7.5 10 % (w/v) PEG 4000 0.05 M Magnesium chloride hexahydrate E01 0 0 0.1 M sodium acetate 4.6 12 % (w/v) PEG 9000 trihydrate E02 0.1 M Lithium sulfate 0.1 M tri-sodium citrate 5.6 12 % (w/v) PEG 4000 monohydrate dihydrate E03 0.1 M Lithium sulfate 0.1 M tri-sodium citrate 5.6 4 % (v/v) PEG 400 monohydrate dihydrate E04 0.1 M Ammonium sulfate 0.1 M HEPES sodium 7.5 0.5 M DI-SODIUM 0.5 M Di-potassium hydrogen salt HYDROGEN phosphate PHOSPHATE E05 0.6 M Magnesium sulfate 0.1 M HEPES sodium 7.5 9 % (v/v) PEG 400 hydrate salt E06 0.1 M Potassium/sodium 0.1 M TRIS HCl 8.5 0.4 M Magnesium tartrate tetrahydrate sulfate hydrate E07 0.2 M Potassium chloride 0.05 M MES 5.6 5 % (w/v) Peg 8000 0.01 M Magnesium chloride hexahydrate E08 0.015 M Magnesium acetate 0.05 M Sodium 6 1.7 M Ammonium 0 0 tetrahydrate cacodylate sulfate E09 0.2 M Potassium chloride 0.05 M Sodium 6.5 10 % (w/v) 1,6-hexanediol 0.005 M Magnesium chloride cacodylate hexahydrate E10 0.01 M Magnesium chloride 0.05 M HEPES sodium 7 4 M Lithium chloride 0 0 hexahydrate salt E11 0.2 M Potassium chloride 0.05 M HEPES sodium 7 20 % (v/v) PEG 200 0.025 M Magnesium sulfate aq. salt E12 0.025 M Magnesium sulfate 0.05 M TRIS HCl 8.5 1.8 M Ammonium 0 0 aq. sulfate F01 0 0 0.1 M Sodium acetate 4.6 1 M Ammonium trihydrate sulfate F02 0.1 M Tri-sodium citrate 0.1 M Tri-sodium 5.6 10 % (v/v) Iso-propanol dihydrate citrate dihydrate F03 0 0 0.1 M ADA 6.5 1 M Ammonium sulfate F04 0.1 M Sodium chloride 0.1 M HEPES sodium 7.5 10 % (w/v) PEG 4000 salt F05 0.6 M Magnesium sulfate 0.1 M HEPES sodium 7.5 4 % (v/v) MPD hydrate salt F06 0 0 0.1 M TRIS HCl 8.5 0.2 M Lithium sulfate monohydrate F07 0.1 M Ammonium sulfate 0.05 M MES 5.6 20 % (w/v) PEG 8000 0.01 M Magnesium chloride hexahydrate F08 0.1 M Potassium chloride 0.05 M Sodium 6 15 % (v/v) Iso-propanol 0.025 M Magnesium chloride cacodylate hexahydrate F09 0.08 M Magnesium acetate 0.05 M Sodium 6.5 15 % (v/v) PEG 400 0 0 tetrahydrate cacodylate F10 0.01 M Magnesium chloride 0.05 M HEPES sodium 7 1.6 M Ammonium 0 0 hexahydrate salt sulfate F11 0.2 M Ammonium acetate 0.05 M HEPES sodium 7 5 % (w/v) PEG 4000 0.15 M Magnesium acetate salt tetrahydrate F12 0.005 M Magnesium sulfate 0.05 M TRIS HCl 8.5 35 % (w/v) 1,6-hexanediol 0 0 aq. G01 0 0 0.1 M Sodium acetate 4.6 1 M Magnesium trihydrate sulfate heptahydrate G02 0.1 M Sodium chloride 0.1 M Tri-sodium 5.6 12 % (v/v) MPD citrate dihydrate G03 0.1 M Lithium sulfate 0.1 M ADA 6.5 12 % (w/v) PEG 4000 2 % (v/v) Iso-propanol monohydrate G04 0.1 M Magnesium chloride 0.1 M HEPES sodium 7.5 18 % (v/v) PEG 400 hexahydrate salt G05 0.1 M Lithium sulfate 0.1 M HEPES sodium 7.5 0.1 M Potassium/ monohydrate salt sodium tartrate tetrahydrate G06 0 0 0.1 M TRIS HCl 8.5 0.5 M Ammonium sulfate G07 0.02 M Magnesium chloride 0.05 M MES 6 15 % (v/v) iso-propanol 0 0 hexahydrate G08 0.04 14 Magnesium chloride 0.05 M Sodium 6 5 % (v/v) MPD 0 0 hexahydrate cacodylate G09 0.2 M Potassium chloride 0.05 M Sodium 6.5 10 % (w/v) PEG 4000 0.01 M Magnesium chloride cacodylate hexahydrate G10 0.005 M Magnesium chloride 0.05 M HEPES sodium 7 25 % (v/v) PEG monomethyl 0 0 hexahydrate salt ether 550 G11 0.1 M Ammonium acetate 0.05 M HEPES sodium 7 5 % (w/v) PEG 8000 0.02 M Magnesium chloride salt hexahydrate G12 0.1 M Potassium chloride 0.05 14 TRIS HCl 8.5 30 % (v/v) PEG 400 0.01 M Magnesium chloride hexahydrate H01 0.1 M Magnesium chloride 0.1 M Sodium acetate 4.6 18 % (v/v) PEG 400 hexahydrate trihydrate H02 0 0 0.1 M Tri-sodium citrate 5.6 1 M Magnesium sulfate dihydrate heptahydrate Di-ammonium H03 0 0 0.1 M ADA 6.5 1 M hydrogen phosphate Potassium/sodium H04 0 0 0.1 M HEPES sodium 7.5 1 M tartrate salt tetrahydrate H05 0.1 M Lithium sulfate 0.1 M TRIS HCl 8.5 12 % (v/v) MPD monohydrate H06 0.1 M Tri-sodium citrate 0.1 M TRIS HCl 8.5 5 % (v/v) PEG 400 dihydrate H07 0.1 M Ammonium acetate 0.05 M MES 6 0.6 M Sodium chloride 0.005 M Magnesium sulfate H08 0.04 M Magnesium acetate 0.05 M Sodium 6 30 % (v/v) MPD 0 0 tetrahydrate cacodylate H09 0.2 M Ammonium acetate 0.05 M Sodium 6.5 10 % (w/v) PEG 4000 0.01 M Calcium chloride cacodylate dihydrate H10 0.2 M Potassium chloride 0.05 M HEPES sodium 7 20% % (w/v) 1,6-hexanediol 0.01 M Magnesium chloride salt hexahydrate H11 0.01 M Magnesium chloride 0.05 M TRIS HCl 7.5 1.6 M Ammonium 0 0 hexahydrate sulfate H12 0.2 M Ammonium chloride 0.05 M TRIS HCl 8.5 30 % (w/v) PEG 4000 0.01 M Calcium chloride dihydrate ANNEXE: ‘Hampton 4” plate Well C U Buffer pH C U Precipitant A01 0.1 M CITRIC ACID 4 0.8 M Ammonium sulfate A02 0.1 M CITRIC ACID 5 0.8 M Ammonium sulfate A03 0.1 M MES 6 0.8 M Ammonium sulfate A04 0.1 M HEPES 7 0.8 M Ammonium sulfate A05 0.1 M TRIS 8 0.8 M Ammonium sulfate A06 0.1 M NONE 9 0.8 M Ammonium sulfate B01 0.1 M CITRIC ACID 4 1.6 M Ammonium sulfate B02 0.1 M CITRIC ACID 5 1.6 M Ammonium sulfate B03 0.1 M MES 6 1.6 M Ammonium sulfate B04 0.1 M HEPES 7 1.6 M Ammonium sulfate B05 0.1 M TRIS 8 1.6 M Ammonium sulfate B06 0.1 M BICINE 9 1.6 M Ammonium sulfate C01 0.1 M CITRIC ACID 4 2.4 M Ammonium sulfate C02 0.1 M CITRIC ACID 5 2.4 M Ammonium sulfate C03 0.1 M MES 6 2.4 M Ammonium sulfate C04 0.1 M HEPES 7 2.4 M Ammonium sulfate C05 0.1 M TRIS 8 2.4 M Ammonium sulfate C06 0.1 M BICINE 9 2.4 M Ammonium sulfate D01 0.1 M CITRIC ACID 4 3 M Ammonium sulfate D02 0.1 M CITRIC ACID 5 3 M Ammonium sulfate D03 0.1 M MES 6 3 M Ammonium sulfate D04 0.1 M HEPES 7 3 M Ammonium sulfate D05 0.1 M TRIS 8 3 M Ammonium sulfate D06 0.1 M BICINE 9 3 M Ammonium sulfate E01 4 1 M Malonate E02 4 1.5 M Malonate E03 4 1.9 M Malonate E04 4 2.4 M Malonate E05 4 2.9 M Malonate E06 4 3.4 M Malonate F01 5 1 M Malonate F02 5 1.5 M Malonate F03 5 1.9 M Malonate F04 5 2.4 M Malonate F05 5 2.9 M MALONATE F06 5 3.4 M MALONATE G01 6 1 M MALONATE G02 6 1.5 M MALONATE G03 6 1.9 M MALONATE G04 6 2.4 M MALONATE G05 6 2.9 M MALONATE G06 6 3.4 M MALONATE H01 7 1 M MALONATE H02 7 1.5 M MALONATE H03 7 1.9 M MALONATE H04 7 2.4 M malonate H05 7 2.9 M malonate H06 7 3.4 M malonate Well C U Buffer pH C U Precipitant C U Additive A07 0.8 M Sodium/potassium 5 0.78 M Sodium 0.016 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate A08 0.8 M Sodium/potassium 5.6 0.72 M Sodium 0.08 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate A09 0.8 M Sodium/potassium 6.3 0.52 M Sodium 0.28 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate A10 0.8 M Sodium/potassium 6.9 0.28 M Sodium 0.52 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate A11 0.8 M Sodium/potassium 7.5 0.12 M Sodium 0.68 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate A12 0.8 M Sodium/potassium 8.2 0.032 M Sodium 0.768 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate B07 1 M Sodium/Potassium 5 0.98 M Sodium 0.02 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate B08 1 M Sodium/potassium 5.6 0.9 M Sodium 0.1 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate B09 1 M Sodium/potassium 6.3 0.65 M Sodium 0.35 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate B10 1 M Sodium/potassium 6.9 0.35 M Sodium 0.65 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate B11 1 M Sodium/potassium 7.5 0.15 M Sodium 0.85 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate B12 1 M Sodium/potassium 8.2 0.04 M Sodium 0.96 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate C07 1.4 M Sodium/potassium 5 1.372 M Sodium 0.028 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate C08 1.4 M Sodium/potassium 5.6 1.26 M Sodium 0.19 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate C09 1.4 M Sodium/potassium 6.3 0.91 M Sodium 0.49 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate C10 1.4 M Sodium/potassium 6.9 0.99 M Sodium 0.91 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate C11 1.4 M Sodium/potassium 7.5 0.21 M Sodium 1.19 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate C12 1.4 M Sodium/potassium 8.2 0.056 M Sodium 1.344 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate D07 1.8 M Sodium/potassium 5 1.764 M Sodium 0.036 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate D08 1.8 M Sodium/potassium 5.6 1.62 M Sodium 0.18 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate D09 1.8 M Sodium/potassium 6.3 1.17 M Sodium 0.63 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate D10 1.8 M Sodium/potassium 6.9 0.63 M Sodium 1.17 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate D11 1.8 M Sodium/potassium 7.5 0.27 M Sodium 1.53 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate D12 1.8 M Sodium/potassium 8.2 0.072 M Sodium 1.728 M Di-potassium phosphate dihydrogen hydrogen phosphate phosphate monohydrate E07 0.1 M CITRIC ACID 4 0.8 M Sodium formate ph4 E08 0.1 M CITRIC ACID 5 0.8 M Sodium formate ph5 E09 0.1 M MES 6 0.8 M Sodium formate ph6 E10 0.1 M HEPES 7 0.8 M Sodium formate ph7 E11 0.1 M TRIS 8 0.8 M Sodium formate ph8 E12 0.1 M BICINE 9 0.8 M Sodium formate ph9 F07 0.1 M CITRIC ACID 4 1.6 M Sodium formate ph4 E08 0.1 M CITRIC ACID 5 1.6 M Sodium formate ph5 F09 0.1 M MES 6 1.6 M Sodium formate ph6 F10 0.1 M HEPES 7 1.6 Fl Sodium formate ph7 F11 0.1 M TRIS 8 1.6 M Sodium formate ph8 F12 0.1 M BICINE 9 1.6 M Sodium formate ph9 G07 0.1 M CITRIC ACID 4 2.4 M Sodium formate ph4 G08 0.1 M CITRIC ACID 5 2.4 M Sodium formate ph5 G09 0.1 M MES 6 2.4 M Sodium formate ph6 G10 0.1 M HEPES 7 2.4 M Sodium formate ph7 G11 0.1 M TRIS 8 2.4 M Sodium formate ph8 G12 0.1 M BICINE 9 2.4 M Sodium formate ph9 H07 0.1 M CITRIC ACID 4 3.2 M Sodium formate ph4 H08 0.1 M CITRIC ACID 5 3.2 M Sodium formate ph5 H09 0.1 M MES 6 3.2 M Sodium formate ph6 H10 0.1 M HEPES 7 3.2 M Sodium formate ph7 H11 0.1 M TRIS 8 3.2 M Sodium formate ph8 H12 0.1 M BICINE 9 3.2 M Sodium formate ph9 Well C U Salt C U Buffer pH C U Precipitant ANNEXE: ‘Hampton 5” plate A01 0.1 M CITRIC ACID 4 5 % (w/v) PEG 6000 A02 0.1 M CITRIC ACID 5 5 % (w/v) PEG 6000 A03 0.1 M MES 6 5 % (w/v) PEG 6000 A04 0.1 M HEPES 7 5 % (w/v) PEG 6000 A05 0.1 M TRIS 8 5 % (w/v) PEG 6000 A06 0.1 M BICINE 9 5 % (w/v) PEG 6000 B01 0.1 M CITRIC ACID 4 10 % (w/v) PEG 6000 B02 0.1 M CITRIC ACID 5 10 % (w/v) PEG 6000 B03 0.1 M MES 6 10 (w/v) PEG 6000 B04 0.1 M HEPES 7 10 % (w/v) PEG 6000 B05 0.1 M TRIS 8 10 % (w/v) PEG 6000 B06 0.1 M BICINE 9 10 % (w/v) PEG 6000 C01 0.1 M CITRIC ACID 4 20 % (w/v) PEG 6000 C02 0.1 M CITRIC ACID 5 20 % (w/v) PEG 6000 C03 0.1 M MES 6 20 % (w/v) PEG 6000 C04 0.1 M HEPES 7 20 % (w/v) PEG 6000 C05 0.1 M TRIS 8 20 % (w/v) PEG 6000 C06 0.1 M BICINE 9 20 % (w/v) PEG 6000 D01 0.1 M CITRIC ACID 4 30 % (w/v) PEG 6000 D02 0.1 M CITRIC ACID 5 30 % (w/v) PEG 6000 D03 0.1 M MES 6 30 % (w/v) PEG 6000 D04 0.1 M HEPES 7 30 % (w/v) PEG 6000 D05 0.1 M TRIS 8 30 % (w/v) PEG 6000 D06 0.1 M BICINE 9 30 % (w/v) PEG 6000 E01 0.1 M CITRIC ACID 4 10 % (v/v) 2-Methyl-2.4-pentanediol E02 0.1 M Sodium acetate 5 10 % (v/v) 2-Methyl-2.4-pentanediol trihydrate E03 0.1 M MES 6 10 % (v/v) 2-Methyl-2.4-pentanediol E04 0.1 M HEPES 7 10 % (v/v) 2-Methyl-2.4-pentanediol E05 0.1 M TRIS 8 10 % (v/v) 2-Methyl-2.4-pentanediol E06 0.1 M BICINE 9 10 % (v/v) 2-Methyl-2.4-pentanediol F01 0.1 M CITRIC ACID 4 20 % (v/v) 2-Methyl-2.4-pentanediol F02 0.1 M Sodium acetate 5 20 % (v/v) 2-Mettlyl-2.4-pentanediol trihydrate F03 0.1 M MES 6 20 % (v/v) 2-Methyl-2.4-pentanediol F04 0.1 M HEPES 7 20 % (v/v) 2-Methyl-2.4-pentanediol F05 0.1 M TRIS 8 20 % (v/v) 2-Methyl-2.4-pentanediol F06 0.1 M BICINE 9 20 % (v/v) 2-Methyl-2.4-pentanediol G01 0.1 M CITRIC ACID 4 40 % (v/v) 2-Methyl-2.4-pentanediol G02 0.1 M Sodium acetate 5 40 % (v/v) 2-Methyl-2.4-pentanediol trihydrate G03 0.1 M MES 6 40 % (v/v) 2-Methyl-2.4-pentanediol G04 0.1 M HEPES 7 40 % (v/v) 2-Methyl-2.4-pentanediol G05 0.1 M TRIS 8 40 % (v/v) 2-Methyl-2.4-pentanediol G06 0.1 M BICINE 9 40 % (v/v) 2-Methyl-2.4-pentanediol H01 0.1 M CITRIC ACID 4 65 % (v/v) 2-Methyl-2.4-pentanediol H02 0.1 M Sodium acetate 5 65 % (v/v) 2-Methyl-2.4-pentanediol trihydrate H03 0.1 M MES 6 65 % (v/v) 2-Methyl-2.4-pentanediol H04 0.1 M HEPES 7 65 % (v/v) 2-Methyl-2.4-pentanediol H05 0.1 M TRIS 8 65 % (v/v) 2-Methyl-2.4-pentanediol H06 0.1 M BICINE 9 65 % (v/v) 2-Methyl-2.4-pentanediol A07 1 M Lithium chloride 0.1 M CITRIC ACID 4 A08 1 M Lithium chloride 0.1 M CITRIC ACID 5 A09 1 M Lithium chloride 0.1 M MES 6 A10 1 M Lithium chloride 0.1 M HEPES 7 A11 1 M Lithium chloride 0.1 M TRIS 8 A12 1 M Lithium chloride 0.1 M BICINE 9 B07 1 M Lithium chloride 0.1 M CITRIC ACID 4 10 % (w/v) PEG 6000 B08 1 M Lithium chloride 0.1 M CITRIC ACID 5 10 % (w/v) PEG 6000 B09 1 M Lithium chloride 0.1 M MES 6 10 % (w/v) PEG 6000 B10 1 M Lithium chloride 0.1 M HEPES 7 10 % (w/v) PEG 6000 B11 1 M Lithium chloride 0.1 M TRIS 8 10 % (w/v) PEG 6000 B12 1 M Lithium chloride 0.1 M BICINE 9 10 % (w/v) PEG 6000 C07 1 M Lithium chloride 0.1 M CITRIC ACID 4 20 % (w/v) PEG 6000 C08 1 M Lithium chloride 0.1 M CITRIC ACID 5 20 % (w/v) PEG 6000 C09 1 M Lithium chloride 0.1 M MES 6 20 % (w/v) PEG 6000 C10 1 M Lithium chloride 0.1 M HEPES 7 20 % (w/v) PEG 6000 C11 1 M Lithium chloride 0.1 M TRIS 8 20 % (w/v) PEG 6000 C12 1 M Lithium chloride 0.1 M BICINE 9 20 % (w/v) PEG 6000 D07 1 M Lithium chloride 0.1 M CITRIC ACID 4 30 % (w/v) PEG 6000 D08 1 M Lithium chloride 0.1 M CITRIC ACID 5 30 % (w/v) PEG 6000 D09 1 M Lithium chloride 0.1 M MES 6 30 % (w/v) PEG 6000 D10 1 M Lithium chloride 0.1 M HEPES 7 30 % (w/v) PEG 6000 D11 1 M Lithium chloride 0.1 M TRIS 8 30 % (w/v) PEG 6000 D12 1 M Lithium chloride 0.1 M BICINE 9 30 % (w/v) PEG 6000 E07 0.1 M CITRIC ACID 4 5 % (v/v) PEG MME 5000 E08 0.1 M CITRIC ACID 5 5 % (v/v) PEG MME 5000 E09 0.1 M MES 6 5 % (v/v) PEG MME 5000 E10 0.1 M HEPES 7 5 % (v/v) PEG MME 5000 E11 0.1 M TRIS 8 5 % (v/v) PEG MME 5000 E12 0.1 M BICINE 9 5 % (v/v) PEG MME 5000 F07 0.1 M CITRIC ACID 4 10 % (v/v) PEG MME 5000 F08 0.1 M CITRIC ACID 5 10 % (v/v) PEG MME 5000 F09 0.1 M MES 6 10 % (v/v) PEG MME 5000 F10 0.1 M HEPES 7 10 % (v/v) PEG MME 5000 F11 0.1 M TRIS 8 10 % (v/v) PEG MME 5000 F12 0.1 M BICINE 9 10 % (v/v) PEG MME 5000 G07 0.1 M CITRIC ACID 4 15 % (v/v) PEG MME 5000 G08 0.1 M CITRIC ACID 5 15 % (v/v) PEG MME 5000 G09 0.1 M MES 6 15 % (v/v) PEG MME 5000 G10 0.1 M HEPES 7 15 % (v/v) PEG MME 5000 G11 0.1 M TRIS 8 15 % (v/v) PEG MME 5000 G12 0.1 M BICINE 9 15 % (v/v) PEG MME 5000 H07 0.1 M CITRIC ACID 4 20 % (v/v) PEG MME 5000 H08 0.1 M CITRIC ACID 5 20 % (v/v) PEG MME 5000 H09 0.1 M MES 6 20 % (v/v) PEG MME 5000 H10 0.1 M HEPES 7 20 % (v/v) PEG MME 5000 H11 0.1 M TRIS 8 20 % (v/v) PEG MME 5000 H12 0.1 M BICINE 9 20 % (v/v) PEG MME 5000 ANNEXE: ‘Hampton 6” plate A01 0.1 M CITRIC ACID 3.5 2 M ammonium sulfate A02 0.1 M BIS-TRIS 5.5 3 M sodium chloride A03 5.6 1.4 M sodium/potassium phosphate A04 7 3.5 M sodium formate pH 7.0 A05 1.1 M Sodium 0.1 M HEPES 7 0.5 % (v/v) jeffamine ED-2001 malonate ph reagent pH 7.0 7.0 A06 0.1 M SODIUM 4.5 25 % (w/v) PEG 3350 ACETATE TRIHYDRATE A07 0.2 M Calcium 0.1 M BIS-TRIS 6.5 45 % (v/v) 2-methyl-2.4-pentanediol chloride A08 0.05 M Ammonium 0.1 M BIS-TRIS 6.5 30 % (v/v) pentaerythritol ethoxylate sulfate (15/4 EO/OH) A09 0.1 M Ammonium 0.1 M BIS-TRIS 5.5 17 % (w/v) PEG 10.000 acetate A10 0.2 M Sodium 0.1 M TRIS 8.5 25 % (w/v) PEG 3350 chloride A11 0.2 M Ammonium 0.1 M TRIS 8.5 25 % (w/v) PEG 3350 acetate A12 0.1 M Succinic acid 15 % (w/v) PEG 3350 ph 7.0 B01 0.1 M Sodium acetate 4.5 2 M ammonium sulfate trihydrate B02 0.1 M BIS-TRIS 6.5 3 M sodium chloride B03 6.9 1.4 M sodium/potassium phosphate B04 7 1.1 M di-ammonium tartrate pH 7.0 B05 1 M Succinic acid 0.1 M HEPES 7 1 % (w/v) PEG MME 2000 ph 7.0 B06 0.1 M BIS-TRIS 5.5 25 % (w/v) PEG 3350 B07 0.2 M Ammonium 0.1 M BIS-TRIS 5.5 45 % (v/v) 2-methyl-2.4- acetate pentanediol B08 0.1 M BIS-TRIS 6.5 45 % (v/v) polypropylene glycol P400 B09 0.2 M Ammonium 0.1 M BIS-TRIS 5.5 25 % (w/v) PEG 3350 sulfate B10 0.2 M Lithium sulfate 0.1 M BIS-TRIS 5.5 25 % (w/v) PEG 3350 B11 0.2 M Magnesium 0.1 M BIS-TRIS 5.5 25 % .(w/v) PEG 3350 chloride B12 0.2 M Sodium 20 % (w/v) PEG 3350 formate C01 0.1 M BIS-TRIS 5.5 2 M ammonium sulfate C02 0.1 M HEPES 7.5 3 M sodium chloride C03 8.2 1.4 M Sodium/potassium phosphate C04 7 2.4 M Sodium malonate ph 7.0 C05 1 M Ammonium sulfate 0.1 M HEPES 7 0.5 % (w/v) PEG 8000 C06 0.1 M BIS-TRIS 6.5 25 % (w/v) PEG 3350 C07 0.2 M Ammonium acetate 0.1 M BIS-TRIS 6.5 45 % (v/v) 2-methyl-2.4-pentanediol Polyacrylic acid 5100 C08 0.02 M Magnesium 0.1 M HEPES 7.5 22 % (w/v) sodium salt chloride C09 0.2 M Ammonium 0.1 M BIS-TRIS 6.5 25 % (w/v) PEG 3350 sulfate C10 0.2 M Lithium sulfate 0.1 M BIS-TRIS 6.5 25 % (w/v) PEG 3350 C11 0.2 M Magnesium 0.1 M BIS-TRIS 6.5 25 % (w/v) PEG 3350 chloride C12 0.15 M DL-malic acid 20 % (w/v) PEG 3350 ph 7.0 D01 0.1 M BIS-TRIS 6.5 2 M Ammonium sulfate D02 0.1 M TRIS 8.5 3 M Sodium chloride D03 0.1 M HEPES 7.5 1.4 M Tri-sodium citrate dihydrate D04 7 35 % (v/v) Tacsimate ph 7.0 D05 15 % (w/v) Tacsimate ph 7.0 0.1 M HEPES 7 2 % (w/v) Peg 3350 D06 0.1 M HEPES 7.5 25 % (w/v) Peg 3350 D07 0.2 M Ammonium 0.1 M HEPES 7.5 45 % (v/v) 2-methyl-2.4-pentanediol acetate D08 0.1 M Cobalt 0.1 M TRIS 8.5 20 % (w/v) Polyvinylpyrrolidone chloride K15 D09 0.2 M Ammonium 0.1 M HEPES 7.5 25 % (w/v) PEG 3350 sulfate ANNEXE: ‘Hampton 6” plate AD- AD- AD- DI- DI- DI- TIVE TIVE Well C U Salt C U Buffer pH C U Precipitant C U TIVE C U 1 C U 2 D10 0.2 M Lithium 0.1 M Hepes 7.5 25 % (w/v) PEG 3350 sulfate D11 0.2 M Magnesium 0.1 M Hepes 7.5 25 % (w/v) PEG 3350 chloride D12 0.1 M Magnesium 15 % (w/v) PEG 3350 formate E01 0.1 M Hepes 7.5 2 M Ammonium sulfate E02 0.1 M Bis-tris 5.5 0.3 M Magnesium formate E03 7 1.8 M Tri-ammonium citrate ph 7.0 E04 7 60 % (w/v) Tacsimateph 7.0 E05 25 % (w/v) PEG 1500 E06 0.1 M Tris 8.5 25 % (w/v) PEG 3350 E07 0.2 M Ammonium 0.1 M Tris 8.5 95 % (v/v) 2-methyl-2.4- acetate pentanediol E08 0.2 M Proline 0.1 M Hepes 7.5 10 % (w/v) PEG 3350 E09 0.2 M Ammonium 0.1 M Tris 8.5 5 % (w/v) PEG 3350 sulfate E10 0.2 M Lithium 0.1 M Tris 8.5 25 % (w/v) PEG 3350 sulfate E11 0.2 M Magnesium 0.1 M Tris 8.5 25 % (w/v) PEG 3350 chloride E12 0.05 M zinc acetate 20 % (w/v) PEG 3350 F01 0.1 M TRIS 8.5 2 M Ammonium sulfate F02 0.1 M BIS-TRIS 6.5 0.5 M Magnesium formate F03 7 0.8 M Succinic acid ph 7.0 F04 0.1 M Sodium chloride 0.1 M BIS-TRIS 6.5 1.5 M Ammonium sulfate F05 0.1 M HEPES 7 30 % (v/v) Jeffamine M- 600 reagent ph 7.0 F06 0.1 M BIS-TRIS 6.5 20 % (w/v) PEG MME 5000 F07 0.05 M Calcium chloride 0.1 M BIS-TRIS 6.5 30 % (v/v) PEG MME 550 F08 0.2 M Trimethylamine 0.1 M TRIS 8.5 20 % (w/v) PEG MME n-oxide 2000 F09 0.2 M Sodium chloride 0.1 M BIS-TRIS 5.5 25 % (w/v) PEG 3350 F10 0.2 M Ammonium acetate 0.1 M BIS-TRIS 5.5 25 % (w/v) PEG 3350 F11 0.2 M Potassium/sodium 20 % (w/v) PEG 3350 tartrate F12 0.2 M Tri-sodium citrate 20 % (w/v) PEG 3350 G01 0.1 M CITRIC 3.5 3 M Sodium ACID chloride G02 0.1 M HEPES 7.5 0.5 M Magnesium formate G03 7 2.1 M DL-malic acid pH 7.0 G04 0.8 M Potassium/sodium 0.1 M TRIS 8.5 0.5 % (w/v) PEG MME tartrate 5000 G05 0.1 M HEPES 7 30 % (v/v) Jeffamine ED- 2001 reagent ph 7.0 G06 0.1 M bis-tris 6.5 28 % (w/v) PEG MME 2000 G07 0.05 M Magnesium 0.1 M HEPES 7.5 30 % (v/v) PEG MME 550 chloride G08 5 % (w/v) Tacsimate ph 0.1 M HEPES 7 10 % (w/v) PEG MME 550 7.0 G09 0.2 M Sodium 0.1 M BIS-TRIS 6.5 25 % (w/v) PEG 3350 chloride G10 0.2 M Ammonium 0.1 M BIS-TRIS 6.5 25 % (w/v) PEG 3350 acetate G11 0.2 M Sodium 20 % (w/v) PEG 3350 malonate ph 7.0 G12 0.1 M Potassium 30 % (w/v) PEG MME 2000 thiocyanate H01 0.1 M Sodium 4.5 3 M Sodium chloride acetate trihydrate H02 0.1 M TRIS 8.5 0.3 M Magnesium formate H03 7 2.8 M Sodium acetate trihydrate ph 7.0 H04 1 M Ammonium 0.1 M BIS-TRIS 5.5 1 % (w/v) PEG 3350 sulfate H05 0.1 M CITRIC ACID 3.5 25 % (w/v) PEG 3350 H06 0.2 M Calcium 0.1 M bis- 5.5 45 % (v/v) 2-methyl-2.4- chloride tris pentanediol H07 0.2 M Potassium 0.05 M HEPES 7.5 30 % (v/v) Pentaerythritol chloride propoxylate (5/4 PO/OH) H08 0.005 M Magnesium 0.1 M HEPES 7.5 12 % (w/v) PEG 3350 0.005 N Nickel 0.005 M Cad- 0.005 M Co- chloride (II) mium balt chlo- Chlo- chlo- ride ride ride H09 0.2 M Sodium 0.1 M HEPES 7.5 25 % (w/v) PEG 3350 chloride H10 0.2 M Ammonium 0.1 M HEPES 7.5 25 %(w/v) PEG 3350 acetate H11 0.2 M Tri- 20 % (w/v) PEG 3350 ammonium citrate ph 7.0 H12 0.15 M Potassium 30 % (w/v) PEG MME bromide 2000

ANNEXE 2B ANNEXE: The “JCSG” plate Well C U Salt C U Buffer pH C U Precipitant C U Precipitant2 D10 0.2 M Calcium acetate 0.1 M Sodium 6.5 40 % (v/v) PEG 300 cacodylate D11 0.14 M Calcium chloride 0.07 M Sodium acetate 4.6 14 % (v/v) Isopropanol 30 % (v/v) Glycerol D12 0.04 M Potassium 16 % (w/v) PEG 8000 20 % (v/v) Glycerol phosphate E01 1 M Tri-sodium 0.1 M Sodium 6.5 citrate cacodylate E02 0.2 M Sodium chloride 0.1 M Sodium 6.5 2 M Ammonium cacodylate sulfate E03 0.2 M Sodium chloride 0.1 M HEPES 7.5 10 % (v/v) Isopropanol E04 0.2 M Lithium sulfate 0.1 M TRIS 8.5 1.26 M Ammonium sulfate E05 0.1 M CAPS 10.5 40 % (v/v) MPD E06 0.2 M Zinc acetate 0.1 M Imidazole 8 20 % (w/v) PEG 3000 E07 0.2 M Zinc acetate 0.1 M Sodium 6.5 10 % (v/v) Isopropanol cacodylate E08 1 M Di-ammonium 0.1 M Sodium acetate 4.5 phosphate E09 1.6 M Magnesium 0.1 M MES 6.5 sulfate E10 0.1 M BICINE 9 10 % (w/v) PEG 6000 E11 0.16 M Calcium acetate 0.08 M Sodium 6.5 14.4 % (w/v) PEG 8000 20 % (v/v) Glycerol cacodylate E12 0.1 M Imidazole 8 10 % (w/v) PEG 8000 F01 0.05 M Cesium chloride 0.1 M MES 6.5 30 % (w/v) Jeffamine M-600 F02 3.15 M Ammonium 0.1 M Tri-sodium 5 sulfate citrate F03 0.1 M TRIS 8 20 % (v/v) MPD F04 0.1 M HEPES 6.5 20 % (w/v) Jeffamine M-600 F05 0.2 M Magnesium 0.1 M TRIS 8.5 50 % (v/v) Ethylene chloride glycol F06 0.1 M BICINE 9 10 % (v/v) MPD F07 0.8 M Succinic acid ph 7.0 F08 2.1 M DL-Malic acid ph 7.0 F09 2.4 M Sodium malonate ph 7.0 F10 1.1 M Sodium malonate 0.1 M HEPES 7 0.5 % (v/v) Jeffamine ED-2001 F11 1 M Succinic acid 0.1 M HEPES 7 1 % (w/v) PEG MME 2000 F12 0.1 M HEPES 7 30 % (v/v) Jeffamine M-600 ph 7.0 G01 0.1 M HEPES 7 30 % (v/v) Jeffamine ED-2001 ph 7.0 G02 0.02 M magnesium 0.1 M HEPES 7.5 22 % (w/v) Polyacrylic chloride acid 5100 sodium salt G03 0.1 M Cobalt chloride 0.1 M TRIS 8.5 20 % (w/v) Polyvinylpyrrolidone K15 G04 0.2 M Trimethylamine 0.1 M TRIS 8.5 20 % (w/v) PEG MME 2000 N-oxide G05 0.005 M Cobalt chloride 0.1 M HEPES 7.5 12 % (w/v) PEG 3350 0.005 M Cadmium chloride G06 0.24 M Sodium 20 % (w/v) PEG 3350 malonate ph 7.0 G07 0.1 M Succinic acid 15 % (w/v) PEG 3350 ph 7.0 G08 0.15 M DL-Malic 20 % (w/v) PEG 3350 acid ph 7.0 G09 0.1 M Potassium 30 % (w/v) PEG MME thiocyanate 2000 G10 0.15 M Potassium 30 % (w/v) PEG MME bromide 2000 G11 2 M Ammonium sulfate 0.1 M BIS-TRIS 5.5 G12 3 M Sodium chloride 0.1 M BIS-TRIS 5.5 H01 0.3 M Magnesium formate 0.1 M BIS-TRIS 5.5 H02 1 M Ammonium sulfate 0.1 M BIS-TRIS 5.5 1 % (w/v) PEG 3350 H03 0.1 M BIS-TRIS 5.5 25 % (w/v) PEG 3350 H04 0.2 M Calcium chloride 0.1 M BIS-TRIS 5.5 45 % (v/v) MPD H05 0.2 M Ammonium acetate 0.1 M BIS-TRIS 5.5 45 % (v/v) MPD H06 0.1 M Ammonium acetate 0.1 M BIS-TRIS 5.5 17 % (w/v) PEG 10.000 H07 0.2 M Ammonium sulfate 0.1 M BIS-TRIS 5.5 25 % (w/v) PEG 3350 H08 0.2 M Sodium chloride 0.1 M BIS-TRIS 5.5 25 % (w/v) PEG 3350 H09 0.2 M Lithium sulfate 0.1 M BIS-TRIS 5.5 25 % (w/v) PEG 3350 H10 0.2 M Ammonium acetate 0.1 M BIS-TRIS 5.5 25 % (w/v) PEG 3350 H11 0.2 M Magnesium chloride 0.1 M BIS-TRIS 5.5 25 % (w/v) PEG 3350 H12 0.2 M Ammonium acetate 0.1 M HEPES 7.5 45 % (v/v) MPD ANNEXE : “The PACT” plate Well C U Salt C U Buffer pH C U Precipitant A01 0 0 0.1 SPG buffer 4 25 % (w/v) PEG 1500 A02 0 0 0.1 SPG buffer 5 25 % (w/v) PEG 1500 A03 0 0 0.1 SPG buffer 6 25 % (w/v) PEG 1500 A04 0 0 0.1 SPG buffer 7 25 % (w/v) PEG 1500 A05 0 0 0.1 SPG buffer 8 25 % (w/v) PEG 1500 A06 0 0 0.1 SPG buffer 9 25 % (w/v) PEG 1500 A07 0.2 Sodium chloride 0.1 Sodium acetate 5 20 % (w/v) PEG 6000 A08 0.2 Ammonium chloride 0.1 Sodium acetate 5 20 % (w/v) PEG 6000 A09 0.2 Lithium chloride 0.1 Sodium acetate 5 20 % (w/v) PEG 6000 A10 0.2 Magnesium chloride 0.1 Sodium acetate 5 20 % (w/v) PEG 6000 A11 0.2 Calcium chloride 0.1 Sodium acetate 5 20 % (w/v) PEG 6000 A12 0.01 Zinc chloride 0.1 Sodium acetate 5 20 % (w/v) PEG 6000 B01 0 0 0.1 MIB buffer 4 25 % (w/v) PEG 1500 B02 0 0 0.1 MIB buffer 5 25 % (w/v) PEG 1500 B03 0 0 0.1 MIB buffer 6 25 % (w/v) PEG 1500 B04 0 0 0.1 MIB buffer 7 25 % (w/v) PEG 1500 B05 0 0 0.1 MIB buffer 8 25 % (w/v) PEG 1500 B06 0 0 0.1 MIB buffer 9 25 % (w/v) PEG 1500 B07 0.2 Sodium chloride 0.1 MES 6 20 % (w/v) PEG 6000 B08 0.2 Ammonium chloride 0.1 MES 6 20 % (w/v) PEG 6000 B09 0.2 Lithium chloride 0.1 MES 6 20 % (w/v) PEG 6000 B10 0.2 Magnesium chloride 0.1 MES 6 20 % (w/v) PEG 6000 B11 0.2 Calcium chloride 0.1 MES 6 20 % (w/v) PEG 6000 B12 0.01 Zinc chloride 0.1 MES 6 20 % (w/v) PEG 6000 C01 0 0 0.1 PCB buffer 4 25 % (w/v) PEG 1500 C02 0 0 0.1 PCB buffer 5 25 % (w/v) PEG 1500 C03 0 0 0.1 PCB buffer 6 25 % (w/v) PEG 1500 C04 0 0 0.1 PCB buffer 7 25 % (w/v) PEG 1500 C05 0 0 0.1 PCB buffer 8 25 % (w/v) PEG 1500 C06 0 0 0.1 PCB buffer 9 25 % (w/v) PEG 1500 C07 0.2 Sodium chloride 0.1 HEPES 7 20 % (w/v) PEG 6000 C08 0.2 Ammonium chloride 0.1 HEPES 7 20 % (w/v) PEG 6000 C09 0.2 Lithium chloride 0.1 HEPES 7 20 % (w/v) PEG 6000 C10 0.2 Magnesium chloride 0.1 HEPES 7 20 % (w/v) PEG 6000 C11 0.2 Calcium chloride 0.1 HEPES 7 20 % (w/v) PEG 6000 C12 0.01 Zinc chloride 0.1 HEPES 7 20 % (w/v) PEG 6000 D01 0 0 0.1 MMT buffer 4 25 % (w/v) PEG 1500 D02 0 0 0.1 MMT buffer 5 25 % (w/v) PEG 1500 D03 0 0 0.1 MMT buffer 6 25 % (w/v) PEG 1500 D04 0 0 0.1 MMT buffer 7 25 % (w/v) PEG 1500 D05 0 0 0.1 MMT buffer 8 25 % (w/v) PEG 1500 D06 0 0 0.1 MMT buffer 9 25 % (w/v) PEG 1500 D07 0.2 Sodium chloride 0.1 TRIS 8 20 % (w/v) PEG 6000 D08 0.2 Ammonium chloride 0.1 TRIS 8 20 % (w/v) PEG 6000 D09 0.2 Lithium chloride 0.1 TRIS 8 20 % (w/v) PEG 6000 D10 0.2 Magnesium chloride 0.1 TRIS 8 20 % (w/v) PEG 6000 D11 0.2 Calcium chloride 0.1 TRIS 8 20 % (w/v) PEG 6000 D12 0.01 Zinc chloride 0.1 TRIS 8 20 % (w/v) PEG 6000 E01 0.2 Sodium fluoride 0 0 0 20 % (w/v) PEG 3350 E02 0.2 Sodium bromide 0 0 0 20 % (w/v) PEG 3350 E03 0.2 Sodium iodide 0 0 0 20 % (w/v) PEG 3350 E04 0.2 Potassium thiocyanate 0 0 0 20 % (w/v) PEG 3350 E05 0.2 Sodium nitrate 0 0 0 20 % (w/v) PEG 3350 E06 0.2 Sodium formate 0 0 0 20 % (w/v) PEG 3350 E07 0.2 Sodium acetate 0 0 0 20 % (w/v) PEG 3350 E08 0.2 Sodium sulphate 0 0 0 20 % (w/v) PEG 3350 E09 0.2 Potassium/ 0 0 0 20 % (w/v) PEG 3350 sodium tartrate E10 0.2 Sodium/potassium 0 0 0 20 % (w/v) PEG 3350 phosphate E11 0.2 Sodium citrate 0 0 0 20 % (w/v) PEG 3350 E12 0.2 Sodium malonate 0 0 0 20 % (w/v) PEG 3350 F01 0.2 Sodium fluoride 0.1 BIS-TRIS 6.5 20 % (w/v) PEG 3350 PROPANE F02 0.2 Sodium bromide 0.1 BIS-TRIS 6.5 20 % (w/v) PEG 3350 PROPANE F03 0.2 Sodium iodide 0.1 BIS-TRIS 6.5 20 % (w/v) PEG 3350 PROPANE F04 0.2 Potassium 0.1 BIS-TRIS 6.5 20 % (w/v) PEG 3350 thiocyanate PROPANE F05 0.2 Sodium nitrate 0.1 BIS-TRIS 6.5 20 % (w/v) PEG 3350 PROPANE F06 0.2 Sodium formate 0.1 BIS-TRIS 6.5 20 % (w/v) PEG 3350 PROPANE F07 0.2 Sodium acetate 0.1 BIS-TRIS 6.5 20 % (w/v) PEG 3350 PROPANE F08 0.2 Sodium sulphate 0.1 BIS-TRIS 6.5 20 % (w/v) PEG 3350 PROPANE F09 0.2 Potassium/ 0.1 BIS-TRIS 6.5 20 % (w/v) PEG 3350 sodium tartrate PROPANE F10 0.2 Sodium/potassium 0.1 BIS-TRIS 6.5 20 % (w/v) PEG 3350 phosphate PROPANE F11 0.2 Sodium citrate 0.1 BIS-TRIS 6.5 20 % (w/v) PEG 3350 PROPANE F12 0.2 Sodium malonate 0.1 BIS-TRIS 6.5 20 % (w/v) PEG 3350 PROPANE G01 0.2 Sodium fluoride 0.1 BIS-TRIS 7.5 20 % (w/v) PEG 3350 PROPANE G02 0.2 Sodium bromide 0.1 BIS-TRIS 7.5 20 % (w/v) PEG 3350 PROPANE G03 0.2 Sodium iodide 0.1 BIS-TRIS 7.5 20 % (w/v) PEG 3350 PROPANE G04 0.2 Potassium 0.1 BIS-TRIS 7.5 0 % (w/v) PEG 3350 thiocyanate PROPANE G05 0.2 Sodium nitrate 0.1 BIS-TRIS 7.5 20 % (w/v) PEG 3350 PROPANE G06 0.2 Sodium formate 0.1 BIS-TRIS 7.5 20 % (w/v) PEG 3350 PROPANE G07 0.2 Sodium acetate 0.1 BIS-TRIS 7.5 20 % (w/v) PEG 3350 PROPANE G08 0.2 Sodium sulphate 0.1 BIS-TRIS 7.5 20 % (w/v) PEG 3350 PROPANE G09 0.2 K/Na tartarte 0.1 BIS-TRIS 7.5 20 % (w/v) PEG 3350 PROPANE G10 0.2 K/Na phosphate 0.1 BIS-TRIS 7.5 20 % (w/v) PEG 3350 PROPANE G11 0.2 Sodium citrate 0.1 BIS-TRIS 7.5 20 % (w/v) PEG 3350 PROPANE G12 0.2 Sodium malonate 0.1 BIS-TRIS 7.5 20 % (w/v) PEG 3350 PROPANE H01 0.2 Sodium fluoride 0.1 BIS-TRIS 8.5 20 % (w/v) PEG 3350 PROPANE H02 0.2 Sodium bromide 0.1 BIS-TRIS 8.5 20 % (w/v) PEG 3350 PROPANE H03 0.2 Sodium iodide 0.1 BIS-TRIS 8.5 20 % (w/v) PEG 3350 PROPANE H04 0.2 Potassium 0.1 BIS-TRIS 8.5 20 % (w/v) PEG 3350 thiocyanate PROPANE H05 0.2 Sodium nitrate 0.1 BIS-TRIS 8.5 20 % (w/v) PEG 3350 PROPANE H06 0.2 Sodium formate 0.1 BIS-TRIS 8.5 20 % (w/v) PEG 3350 PROPANE H07 0.2 Sodium acetate 0.1 BIS-TRIS 8.5 20 % (w/v) PEG 3350 PROPANE H08 0.2 Sodium sulphate 0.1 BIS-TRIS 8.5 20 % (w/v) PEG 3350 PROPANE H09 0.2 K/Na tartrate 0.1 BIS-TRIS 8.5 20 % (w/v) PEG 3350 PROPANE H10 0.2 K/Na phosphate 0.1 BIS-TRIS 8.5 20 % (w/v) PEG 3350 PROPANE H11 0.2 Sodium citrate 0.1 BIS-TRIS 8.5 20 % (w/v) PEG 3350 PROPANE H12 0.2 Sodium malonate 0.1 BIS-TRIS 8.5 20 % (w/v) PEG 3350 PROPANE ANNEXE: Qiagen “Classic suite” plate Well C U Salt C U Buffer pH C U Main Precipitant C U Additive A03 0.2 M Magnesium chloride 0.1 M TRIS 8.5 3.4 M 1.6-hexanediol A02 0.1 M Tri-sodium citrate 5.6 2.5 M 1.6-hexanediol A01 0.01 M Cobalt chloride 0.1 M Sodium acetate 4.6 1 M 1.6-hexanediol C04 0.1 M HEPES 7.5 2 M Ammonium formate C03 0.1 M TRIS HCl 8.5 2 M Ammonium phosphate C02 0.1 M Tri-sodium citrate 5.6 1 M Ammonium phosphate C01 0.4 M Ammonium phosphate C08 0.1 M Sodium chloride 0.1 M HEPES 7.5 1.6 M Ammonium sulfate C09 0.01 M Cobalt chloride 0.1 M MES 6.5 1.8 M Ammonium sulfate C05 0 0 0 0.1 M sodium acetate 4.6 2 M Ammonium sulfate C06 0 0 0 0.1 M TRIS HCl 8.5 2 M Ammonium sulfate C10 0.2 M K/Na tartrate 0.1 M Tri-sodium citrate 5.6 2 M Ammonium sulfate C07 2 M Ammonium sulfate E06 0.5 M Sodium chloride 0.01 M Ctab 0.01 M Magnesium chloride E02 0.1 M MES 6.5 10 % (v/v) Dioxane 1.6 M Ammonium sulfate E03 0 0 0 0 35 % (v/v) Dioxane E01 0.1 M BICINE 9 2 % (v/v) Dioxane 10 % (w/v) PEG 20000 A12 0 0 0 0 10 % (v/v) Ethanol 1.5 M Sodium chloride B01 0.1 M TRIS 8.5 20 % (v/v) Ethanol B02 25 % (v/v) Ethylene glycol E04 0.5 M Sodium chloride 0.1 M Tri-sodium citrate 5.6 2 % (v/v) Ethylene imine polymer E05 0.1 M TRIS 8.5 12 % (v/v) Glycerol 1.5 M Ammonium sulfate C11 1 M Imidazole ph 7.0 A10 0.2 M Magnesium chloride 0.1 M HEPES sodium salt 7.5 30 % (v/v) Isopropanol A08 0.2 M Tri-sodium citrate 0.1 M HEPES sodiums alt 7.5 20 % (v/v) Isopropanol A05 0.1 M HEPES sodium salt 7.5 10 % (v/v) Isopropanol 20 % (w/v) PEG 4000 A06 0.2 M Calcium chloride 0.1 M Sodium acetate 4.6 20 % (v/v) Isopropanol A09 0.2 M Tri-sodium citrate 0.1 M Sodium cacodylate 6.5 30 % (v/v) Isopropanol A11 0.2 M Ammonium acetate 0.1 M TRIS HCl 8.5 30 % (v/v) Isopropanol A07 0.1 M Tri-sodium citrate 5.6 20 % (v/v) Isopropanol 20 % (w/v) PEG 4000 A04 0 0 0 0 5 % (v/v) Isopropanol 2 M Ammonium sulfate E08 0.1 M HEPES 7.5 20 % (v/v) Jeffamine M-600 E07 0.01 M Ferric chloride 0.1 M Tri-sodium citrate 5.6 10 % (v/v) Jeffamine M-600 D01 0.1 M HEPES sodium salt 7.5 0.8 M Potassium/ sodium tartrate C12 0.4 M Potassium/ sodium tartrate E11 0.1 M HEPES sodium salt 7.5 1.5 M Lithium sulfate E10 0.01 M Nickel chloride 0.1 M TRIS 8.5 1 M Lithium sulfate E11 0.5 M Ammonium sulfate 0.1 M Tri-sodium citrate 5.6 1 M Lithium sulfate E12 0.1 M BICINE 9 2 M Magnesium chloride F01 0.2 M Magnesium formate F02 0.1 M MES 6.5 1.6 M Magnesium sulfate B10 0.1 M HEPES 7.5 70 % (v/v) MPD B07 0.2 M Tri-sodium citrate 0.1 M HEPES sodium salt 7.5 30 % (v/v) MPD B03 0.02 M Calcium chloride 0.1 M Sodium acetate 4.6 30 % (v/v) MPD B04 0.2 M Sodium chloride 0.1 M Sodium acetate 4.6 30 % (v/v) MPD B09 0.2 M Ammonium phosphate 0.1 M TRIS 8.5 50 % (v/v) MPD B05 0.2 M Ammonium acetate 0.1 M Tri-sodium citrate 5.6 30 % (v/v) MPD B08 0.5 M Ammonium sulfate 0.1 M HEPES 7.5 30 % (v/v) MPD B06 0.2 M Magnesium acetate 0.1 M Sodium cacodylate 6.5 30 % (v/v) MPD G08 10 % (w/v) PEG 1000 10 % (w/v) PEG 8000 H11 0.1 M HEPES 7.5 20 % (w/v) PEG 10000 8 % (v/v) Ethylene glycol G09 30 % (w/v) PEG 1500 G11 0.2 M Ammonium sulfate 0.1 M Sodium acetate 4.6 30 % (w/v) PEG 2000 MME G10 0.01 M Nickel chloride 0.1 M TRIS 8.5 20 % (w/v) PEG 2000 MME H12 0.1 M MES 6.5 12 % (w/v) PEG 20000 G02 0.2 M Calcium chloride 0.1 M HEPES sodium salt 7.5 28 % (v/v) PEG 400 G04 0.2 M Magnesium chloride 0.1 M HEPES sodium salt 7.5 30 % (v/v) PEG 400 G05 0.2 M Tri-sodium citrate 0.1 M TRIS HCl 8.5 30 % (v/v) PEG 400 G01 0.1 M HEPES sodium salt 7.5 2 % (v/v) PEG 400 2 M Ammonium sulfate G03 0.1 M Cadmium chloride 0.1 M Sodium acetate 4.6 30 % (v/v) PEG 400 H02 0.2 M Ammonium acetate 0.1 M Sodium acetate 4.6 30 % (w/v) PEG 4000 H01 0.2 M Ammonium sulfate 0.1 M Sodium acetate 4.6 25 % (w/v) PEG 4000 H05 0.2 M Lithium sulfate 0.1 M TRIS HCl 8.5 30 % (w/v) PEG 4000 H06 0.2 M Sodium acetate 0.1 M TRIS HCl 8.5 30 % (w/v) PEG 4000 H03 0.2 M Ammonium acetate 0.1 M Tri-sodium citrate 5.6 30 % (w/v) PEG 4000 H07 0.2 M Ammonium sulfate 30 % (w/v) PEG 4000 G12 0.1 M Sodium acetate 4.6 8 % (w/v) PEG 4000 H04 0.2 M Magnesium chloride 0.1 M TRIS HCl 8.5 30 % (w/v) PEG 4000 H08 0.2 M Ammonium sulfate 0.1 M MES 6.5 30 % (w/v) PEG 5000 MME G06 0.1 M Sodium chloride 0.1 M bicine 9 20 % (w/v) PEG 550 MME G07 0.01 M Zinc sulfate 0.1 M MES 6.5 25 % (w/v) PEG 550 MME H09 0.1 M HEPES 7.5 10 % (w/v) PEG 6000 5 % (v/v) MPD H10 10 % (w/v) PEG 6000 2 M sodium chloride F04 0.1 M HEPES 7.5 10 % (w/v) PEG 8000 F06 0.2 M Zinc acetate 0.1 M Sodium cacodylate 6.5 18 % (w/v) PEG 8000 F10 0.2 M Ammonium sulfate 0.1 M Sodium cacodylate 6.5 30 % (w/v) PEG 8000 F07 0.2 M Calcium acetate 0.1 M Sodium cacodylate 6.5 18 % (w/v) PEG 8000 F08 0.2 M Magnesium acetate 0.1 M Sodium cacodylate 6.5 20 % (w/v) PEG 8000 F11 0.2 M Sodium acetate 0.1 M Sodium cacodylate 6.5 30 % (w/v) PEG 8000 F12 0.2 M Ammonium sulfate 30 % (w/v) PEG 8000 F05 0.5 M Lithium sulfate 15 % (w/v) PEG 8000 F09 0.05 M Potassium phosphate 20 % (w/v) PEG 8000 F03 0.1 M TRIS HCl 8.5 8 % (w/v) PEG 8000 D03 0.05 M Cadmium sulfate 0.1 M HEPES 7.5 1 M Sodium acetate D02 0.1 M Imidazole 6.5 1 M Sodium acetate D04 0.1 M Sodium cacodylate 6.5 1.4 M Sodium acetate D07 0.1 M HEPES 7.5 4.3 M Sodium chloride D06 0.1 M Sodium phosphate 0.1 M MES 6.5 2 M Sodium chloride 0.1 M potassium phosphate D05 0.1 M Sodium acetate 4.6 2 M Sodium chloride D11 0.1 M Sodium acetate 4.6 2 M Sodium formate D12 4 M Sodium formate D10 0.1 M HEPES sodium salt 7.5 0.8 M Sodium phosphate 0.8 M potassium phosphate B11 0.1 M TRIS 8.5 25 % (v/v) tert-butanol B12 0.1 M Tri-sodium citrate 5.6 35 % (v/v) tert-butanol D08 0.1 M HEPES sodium salt 7.5 1.4 M tri-sodium citrate D09 6.5 1.6 M tri-sodium citrate pH 6.5 ANNEXE : Qiagen “The PEGS” plate Well C U Salt C U Buffer pH C U Precipitant A01 0.1 M Sodium acetate 4.6 40 % (v/v) PEG 200 A02 0.1 M Sodium acetate 30 % (v/v) PEG 300 A03 0.1 M Sodium acetate 4.6 30 % (v/v) PEG 400 A04 0.1 M Sodium acetate 4.6 25 % (v/v) PEG 550 MME A05 0.1 M Sodium acetate 4.6 25 % (w/v) PEG 1000 A06 0.1 M Sodium acetate 9.6 25 % (w/v) PEG 2000 MME A07 0.1 M MES 6.5 40 % (v/v) PEG 200 A08 0.1 M MES 6.5 30 % (v/v) PEG 300 A09 0.1 M MES 6.5 30 % (v/v) PEG 400 A10 0.1 M MES 6.5 25 % (v/v) PEG 550 MME A11 0.1 M MES 6.5 25 % (w/v) PEG 1000 A12 0.1 M MES 6.5 25 % (w/v) PEG 2000 MME B01 0.1 M HEPES sodium salt 7.5 40 % (v/v) PEG 200 B02 0.1 M HEPES sodium salt 7.5 30 % (v/v) PEG 300 B03 0.1 M HEPES sodium salt 7.5 30 % (v/v) PEG 400 B04 0.1 M HEPES sodium salt 7.5 25 % (v/v) PEG 550 MME B05 0.1 M HEPES sodium salt 7.5 25 % (w/v) PEG 1000 B06 0.1 M HEPES sodium salt 7.5 25 % (w/v) PEG 2000 MME B07 0.1 M TRIS HCl 8.5 40 % (v/v) PEG 200 B08 0.1 M TRIS HCl 8.5 30 % (v/v) PEG 300 B09 0.1 M TRIS HCl 8.5 30 % (v/v) PEG 400 B10 0.1 M TRIS HCl 8.5 25 % (v/v) PEG 550 MME B11 0.1 M TRIS HCl 8.5 25 % (w/v) PEG 1000 B12 0.1 M TRIS HCl 8.5 25 % (w/v) PEG 2000 MME C01 0.1 M Sodium acetate 4.6 25 % (w/v) PEG 3000 C02 0.1 M Sodium acetate 4.6 25 % (w/v) PEG 4000 C03 0.1 M Sodium acetate 9.6 25 % (w/v) PEG 6000 C04 0.1 M Sodium acetate 9.6 25 % (w/v) PEG 8000 C05 0.1 M Sodium acetate 9.6 20 % (w/v) PEG 10000 C06 0.1 M Sodium acetate 9.6 15 % (w/v) PEG 20000 C07 0.1 M MES 6.5 25 % (w/v) PEG 3000 C08 0.1 M MES 6.5 25 % (w/v) PEG 4000 C09 0.1 M MES 6.5 25 % (w/v) PEG 6000 C10 0.1 M MES 6.5 25 % (w/v) PEG 8000 C11 0.1 M MES 6.5 20 % (w/v) PEG 10000 C12 0.1 M MES 6.5 15 % (w/v) PEG 20000 D01 0.1 M HEPES sodium salt 7.5 25 % (w/v) PEG 3000 D02 0.1 M HEPES sodium salt 7.5 25 % (w/v) PEG 4000 D03 0.1 M HEPES sodium salt 7.5 25 % (w/v) PEG 6000 D04 0.1 M HEPES sodium salt 7.5 25 % (w/v) PEG 8000 D05 0.1 M HEPES sodium salt 7.5 20 % (w/v) PEG 10000 D06 0.1 M HEPES sodium salt 7.5 15 % (w/v) PEG 20000 D07 0.1 M TRIS HCl 7.5 25 % (w/v) PEG 3000 D08 0.1 M TRIS HCl 8.5 25 % (w/v) PEG 4000 D09 0.1 M TRIS HCl 8.5 25 % (w/v) PEG 6000 D10 0.1 M TRIS HCl 8.5 25 % (w/v) PEG 8000 D11 0.1 M TRIS HCl 8.5 20 % (w/v) PEG 10000 D12 0.1 M TRIS HCl 8.5 15 % (w/v) PEG 20000 E01 0.2 M Sodium fluoride 20 % (w/v) PEG 3350 E02 0.2 M Potassium fluoride 20 % (w/v) PEG 3350 E03 0.2 M Ammonium fluoride 20 % (w/v) PEG 3350 E04 0.2 M Lithium chloride 20 % (w/v) PEG 3350 E05 0.2 M Magnesium chloride 20 % (w/v) PEG 3350 E06 0.2 M Sodium chloride 20 % (w/v) PEG 3350 E07 0.2 M Calcium chloride 20 % (w/v) PEG 3350 E08 0.2 M Potassium chloride 20 % (w/v) PEG 3350 E09 0.2 M Ammonium chloride 20 % (w/v) PEG 3350 E10 0.2 M Sodium iodide 20 % (w/v) PEG 3350 E11 0.2 M Potassium iodide 20 % (w/v) PEG 3350 E12 0.2 M Ammonium iodide 20 % (w/v) PEG 3350 F01 0.2 M Sodium 20 % (w/v) PEG 3350 thiocyanate F02 0.2 M Potassium 20 % (w/v) PEG 3350 thiocyanate F03 0.2 M Lithium nitrate 20 % (w/v) PEG 3350 F04 0.2 M Magnesium nitrate 20 % (w/v) PEG 3350 F05 0.2 M Sodium nitrate 20 % (w/v) PEG 3350 F06 0.2 M Potassium nitrate 20 % (w/v) PEG 3350 F07 0.2 M Ammonium nitrate 20 % (w/v) PEG 3350 F08 0.2 M Magnesium formate 20 % (w/v) PEG 3350 F09 0.2 M Sodium formate 20 % (w/v) PEG 3350 F10 0.2 M Potassium formate 20 % (w/v) PEG 3350 F11 0.2 M Ammonium formate 20 % (w/v) PEG 3350 F12 0.2 M Lithium acetate 20 % (w/v) PEG 3350 G01 0.2 M Magnesium acetate 20 % (w/v) PEG 3350 G02 0.2 M Zinc acetate 20 % (w/v) PEG 3350 G03 0.2 M Sodium acetate 20 % (w/v) PEG 3350 G04 0.2 M Calcium acetate 20 % (w/v) PEG 3350 G05 0.2 M Potassium acetate 20 % (w/v) PEG 3350 G06 0.2 M Ammonium acetate 20 % (w/v) PEG 3350 G07 0.2 M Lithium sulfate 20 % (w/v) PEG 3350 G08 0.2 M Magnesium sulfate 20 % (w/v) PEG 3350 G09 0.2 M Sodium sulfate 20 % (w/v) PEG 3350 G10 0.2 M Potassium sulfate 20 % (w/v) PEG 3350 G11 0.2 M Ammonium sulfate 20 % (w/v) PEG 3350 G12 0.2 M Di-sodium tartrate 20 % (w/v) PEG 3350 H01 0.2 M Potassium/ 20 % (w/v) PEG 3350 sodium tartrate H02 0.2 M Di-ammonium 20 % (w/v) PEG 3350 tartrate H03 0.2 M Sodium phosphate 20 % (w/v) PEG 3350 H04 0.2 M Di-sodium 20 % (w/v) PEG 3350 phosphate H05 0.2 M Potassium 20 % (w/v) PEG 3350 phosphate H06 0.2 M Di-potassium 20 % (w/v) PEG 3350 phosphate H07 0.2 M Ammonium 20 % (w/v) PEG 3350 phosphate H08 0.2 M Di-ammonium 20 % (w/v) PEG 3350 phosphate H09 0.2 M Tri-lithium citrate 20 % (w/v) PEG 3350 H10 0.2 M Tri-sodium citrate 20 % (w/v) PEG 3350 H11 0.2 M Tri-potassium 20 % (w/v) PEG 3350 citrate H12 0.18 M Tri-ammonium 20 % (w/v) PEG 3350 citrate ANNEXE: Rigaku “Wizard I & II” plate Well C U Salt C U Buffer pH C U Precipitant C U ADDITIVE D10 0.2 M Calcium acetate 0.1 M Imidazole/HCl 8 10 % (w/v) PEG-8000 D11 0.2 M Lithium sulfate 0.1 M TRIS BASE/HCl 8.5 1.26 M Ammonium sulfate D12 0.2 M Zinc acetate 0.1 M Sodium acetate/ 4.5 20 % (w/v) PEG-1000 citric acid E01 0.2 M Zinc acetate 0.1 M Sodium acetate/ 4.5 10 % (w/v) PEG-3000 citric acid E02 0.2 M Lithium sulfate 0.1 M MES/NaOH 6 3.5 % (v/v) 2-methyl-2,4-pentanediol E03 0.2 M Magnesium chloride 0.1 M Tris base/HCl 8.5 20 % (w/v) PEG-8000 E04 0.2 M Sodium chloride 0.1 M sodium cacodylate/ 6.5 2 M Ammonium sulfate . HCl E05 0.2 M Sodium chloride 0.1 M HEPES/NaOH 7.5 20 % (v/v) 1,4-butanediol E06 0.2 M Lithium sulfate 0.1 M Sodium phosphate 4.2 10 % (v/v) 2-propanol dibasic/citric acid E07 0.2 M Sodium chloride 0.1 M TRIS BASE/HCl 7 30 % (w/v) PEG-3000 E08 0.2 M Sodium chloride 0.1 M Potassium 6.2 10 % (w/v) PEG-8000 phosphate monobasic/sodium phosphate dibasic E09 0.1 M Sodium phosphate 4.2 2 M Ammonium sulfate dibasic/citric acid E10 0.1 M TRIS BASE/HCL 8.5 1 M Ammonium phosphate dibasic E11 0.2 M Zinc acetate 0.1 M Sodium cacodylate/ 6.5 10 % (v/v) 2-propanol HCl E12 0.2 M Lithium sulfate 0.1 M Sodium cacodylate/ 6.5 30 % (v/v) PEG-400 HCl F01 0.2 M Lithium sulfate 0.1 M Sodium citrate/ 5.5 15 % (v/v) Reagent alcohol* citric acid F02 0.2 M Sodium chloride 0.1 M Potassium 6.2 20 % (w/v) PEG-1000 phosphate monobasic/sodium phosphate dibasic F03 0.1 M HEPES/NaOH 7.5 1.26 M Ammonium sulfate F04 0.1 M CHES/NaOH 9.5 1 M Sodium citrate tribasic F05 0.2 M Magnesium chloride 0.1 M TRIS BASE/HCl 7 2.5 M Sodium chloride F06 0.2 M Calcium acetate 0.1 M TRIS BASE/HCl 7 20 % (w/v) PEG-3000 F07 0.1 M Sodium phosphate 4.2 1.6 M Sodium phosphate 0.4 M potassium dibasic/citric acid monobasic phosphate dibasic F08 0.2 M Zinc acetate 0.1 M MES/NaOH 6 15 % (v/v) reagent alcohol* F09 0.1 M Sodium acetate/ 4.5 35 % (v/v) 2-methyl-2,4-pentanediol citric acid F10 0.1 M Imidazole/HCl 8 10 % (v/v) 2-propanol F11 0.2 M Magnesium chloride 0.1 M HEPES/NaOH 7.5 15 % (v/v) Reagent alcohol* F12 0.2 M Sodium chloride 0.1 M Imidazole/HCl 8 30 % (v/v) PEG-8000 G01 0.2 M Sodium chloride 0.1 M HEPES/NaOH 7.5 35 % (v/v) 2-methyl-2,4-pentanediol G02 0.1 M CHES/NaOH 9.5 30 % (v/v) PEG-400 G03 0.2 M Magnesium chloride 0.1 M sodium cacodylate/ 6.5 10 % (w/v) PEG-3000 HCl G04 0.2 M Calcium acetate 0.1 M MES/NaOH 6 20 % (w/v) PEG-8000 G05 0.2 M Sodium chloride 0.1 M CHES/NaOH 9.5 26 M Ammonium sulfate G06 0.2 M Zinc acetate 0.1 M Imidazole/HCl 8 20 % (v/v) 1,4-butanediol G07 0.2 M Sodium chloride 0.1 M TRIS BASE/HCL 7 1 M Sodium citrate tribasic G08 0.1 M TRIS BASE/HCl 8.5 20 % (w/v) PEG-1000 G09 0.2 M Sodium chloride 0.1 M Sodium citrate/ 5.5 1 M Ammonium citric acid phosphate dibasic G10 0.1 M Imidazole/HCl 8 10 % (w/v) PEG-8000 G11 0.1 M Sodium acetate/ 4.5 0.8 M Sodium phosphate 1.2 M Potassium citric acid monobasic phosphate dibasic G12 0.2 M Sodium chloride 0.1 M Sodium phosphate 4.2 10 % (w/v) PEG-3000 dibasic/citric acid H01 0.2 M Lithium sulfate 0.1 M TRIS BASE/HCl 7 1 M Potassium/ sodium tartrate H02 0.2 M Lithium sulfate 0.1 M Sodium acetate/ 4.5 2.5 M Sodium chloride citric acid H03 0.2 M Sodium chloride 0.1 M CAPS/NaOH 10.5 20 % (w/v) PEG-8000 H04 0.2 M Zinc acetate 0.1 M Imidazole/HCl 8 20 % (w/v) PEG-3000 H05 0.2 M Lithium sulfate 0.1 M TRIS BASE/HCl 7 2 M Ammonium sulfate H06 0.2 M Sodium chloride 0.1 M HEPES/NaOH 7.5 30 % (v/v) PEG-400 H07 0.2 M Magnesium chloride 0.1 M TRIS BASE/HCl 7 10 % (w/v) PEG-8000 H08 0.2 M Magnesium chloride 0.1 M Sodium cacodylate/ 6.5 20 % (w/v) PEG-1000 HCl H09 0.1 M MES/NaOH 6 1.26 M Ammonium sulfate H10 0.2 M Sodium chloride 0.1 M Imidazole/HCl 8 1 M Ammonium phosphate dibasic H11 0.2 M Zinc acetate 0.1 M Imidazole/HCl 8 2.5 M Sodium chloride H12 0.1 M MES/NaOH 6 1 M Potassium/ sodium tartrate A01 0.1 M CHES/NaOH 9.5 20 % (w/v) PEG-8000 A02 0.2 M Sodium chloride 0.1 M HEPES/NaOH 7.5 10 % (v/v) 2-propanol A03 0.1 M CHES/NaOH 9.5 15 % (v/v) reagent alcohol* A04 0.2 M Magnesium chloride 0.1 M Imidazole/HCl 8 35 % (v/v) 2-methyl-2.4- pentanediol A05 0.1 M CAPS/NaOH 10.5 30 % (v/v) PEG-400 A06 0.1 M Sodium citrate/ 5.5 20 % (w/v) PEG-3000 citric acid A07 0.2 M Zinc acetate 0.1 M MES/NaOH 6 10 % (w/v) PEG-8000 A08 0.1 M Sodium citrate/ 5.5 2 M ammonium sulfate citric acid A09 0.1 M Sodium acetate/ 4.5 1 M ammonium citric acid phosphate dibasic A10 0.1 M TRIS BASE/HCl 7 20 % (w/v) PEG-2000 MME A11 0.2 M Lithium sulfate 0.1 M MES/NaOH 6 20 % (v/v) 1.4-butanediol A12 0.2 M Calcium acetate 0.1 M Imidazole/HCl 8 20 % (w/v) PEG-1000 B01 0.1 M Sodium cacodylate/ 6.5 1.26 M ammonium sulfate HCl B02 0.1 M Sodium cacodylate/ 6.5 1 M sodium citrate tribasic HCl B03 0.2 M Lithium sulfate 0.1 M Imidazole/HCl 8 10 % (w/v) PEG-3000 B04 0.1 M Potassium 6.2 2.5 M Sodium chloride phosphate monobasic/sodium phosphate dibasic B05 0.2 M Lithium sulfate 0.1 M Sodium acetate/ 4.5 30 % (w/v) PEG-8000 citric acid B06 0.2 M Sodium chloride 0.1 M Imidazole/HCl 8 1 M Potassium/ sodium tartrate B07 0.1 M TRIS BASE/HCl 7 20 % (w/v) PEG-1000 B08 0.2 M Sodium chloride 0.1 M Imidazole/HCl 8 0.4 M Sodium phosphate 1.6 M Potassium monobasic phosphate B09 0.1 M HEPES/NaOH 7.5 20 % (w/v) PEG-8000 dibasic B10 0.1 M TRIS BASE/HCl 8.5 10 % (v/v) 2-propanol B11 0.2 M Magnesium chloride 0.1 M Imidazole/HCl 8 15 % (v/v) Reagent alcohol* B12 0.2 M Sodium chloride 0.1 M TRIS BASE/HCl 7 35 % (v/v) 2-methyl-2.4- pentanediol C01 0.2 M Magnesium chloride 0.1 M TRIS BASE/HCl 8.5 30 % (v/v) PEG-400 C02 0.1 M CHES/NaOH 9.5 10 % (w/v) PEG-3000 C03 0.2 M Lithium sulfate 0.1 M CAPS/NaOH 10.5 1.2 M Sodium phosphate 0.8 M Potassium monobasic phosphate dibasic C04 0.2 M Sodium chloride 0.1 M HEPES/NaOH 7.5 20 % (w/v) PEG-3000 C05 0.2 M Sodium chloride 0.1 M CHES/NaOH 9.5 10 % (w/v) PEG-8000 C06 0.2 M Sodium chloride 0.1 M Sodium acetate/ 4.5 1.26 M Ammonium sulfate citric acid C07 0.2 M Sodium chloride 0.1 M Sodium phosphate 4.2 20 % (w/v) PEG-8000 dibasic/citric acid C08 0.1 M Potassium 6.2 10 % (w/v) PEG-3000 phosphate monobasic/sodium phosphate dibasic C09 0.2 M Lithium sulfate 0.1 M CAPS/NaOH 10.5 2 M Ammonium sulfate C10 0.1 M Imidazole/HCl 8 1 M Ammonium phosphate dibasic C11 0.1 M Sodium acetate/ 4.5 20 % (v/v) 1.4-butanediol citric acid C12 0.1 M Imidazole/HCl 8 1 M Sodium citrate tribasic D01 0.1 M Imidazole/HCl 8 2.5 M Sodium chloride D02 0.2 M Lithium sulfate 0.1 M CHES/NaOH 9.5 1 M Potassium/ sodium tartrate D03 0.2 M Lithium sulfate 0.1 M Sodium phosphate 4.2 20 % (w/v) PEG-1000 dibasic/citric acid D04 0.2 M Calcium acetate 0.1 M MES/NaOH 6 10 % (v/v) 2-propanol D05 0.1 M CHES/NaOH 9.5 30 % (w/v) PEG-3000 D06 0.1 M TRIS BASE/HCl 7 15 % (v/v) Reagent alcohol* D07 0.1 M Potassium 6.2 35 % (v/v) 2-methyl-2.4- phosphate monobasic/sodium pentanediol phosphate dibasic D08 0.2 M Calcium acetate 0.1 M Sodium acetate/ 4.5 30 % (v/v) PEG-400 citric acid D09 0.1 M Sodium acetate/ 4.5 20 % (w/v) PEG-3000 citric acid ANNEXE: Hampton “Salt grid” plate Well C U Buffer pH C U Precipitant C U Precipitant 2 pH A01 0.1 M CITRIC ACID 4 0.8 M Ammonium sulfate A02 0.1 M CITRIC ACID 5 0.8 M Ammonium sulfate A03 0.1 M MES monohydrate 6 0.8 M Ammonium sulfate A04 0.1 M HEPES 7 0.8 M Ammonium sulfate A05 0.1 M TRIS 8 0.8 M Ammonium sulfate A06 0.1 M BICINE 9 0.8 M Ammonium sulfate 801 0.1 M CITRIC ACID 4 1.6 M Ammonium sulfate 802 0.1 M CITRIC ACID 5 1.6 M Ammonium sulfate B03 0.1 M MES monohydrate 6 1.6 M Ammonium sulfate B04 0.1 M HEPES 7 1.6 M Ammonium sulfate 805 0.1 M TRIS 8 1.6 M Ammonium sulfate B06 0.1 M BICINE 9 1.6 M Ammonium sulfate C01 0.1 M CITRIC ACID 4 2.4 M Ammonium sulfate C02 0.1 M CITRIC ACID 5 2.4 M Ammonium sulfate C03 0.1 M MES monohydrate 6 2.4 M Ammonium sulfate C04 0.1 M HEPES 7 2.4 M Ammonium sulfate C05 0.1 M TRIS 8 2.4 M Ammonium sulfate C06 0.1 M BICINE 9 2.4 M Ammonium sulfate D01 0.1 M CITRIC ACID 4 3 M Ammonium sulfate D02 0.1 M CITRIC ACID 5 3 M Ammonium sulfate D03 0.1 M MES monohydrate 6 3 M Ammonium sulfate D04 0.1 M HEPES 7 3 M Ammonium sulfate D05 0.1 M TRIS 8 3 M Ammonium sulfate D06 0.1 M BICINE 9 3 M Ammonium sulfate E01 4 1 M Malonate E02 4 1.5 M Malonate E03 4 1.9 M Malonate E04 4 2.4 M Malonate E05 9 2.9 M Malonate E06 4 3.4 M Malonate F01 5 1 M Malonate F02 5 1.5 M Malonate F03 5 1.9 M Malonate F04 5 2.4 M Malonate F05 5 2.9 M Malonate F06 5 3.4 M Malonate G01 6 1 M Malonate G02 6 1.5 M Malonate G03 6 1.9 M Malonate G04 6 2.4 M Malonate G05 6 2.9 M Malonate G06 6 3.4 M Malonate H01 7 1 M Malonate H02 7 1.5 M Malonate H03 7 1.9 M Malonate H04 7 2.4 M Malonate 0 0 0 0 H05 7 2.9 M Malonate 0 0 0 0 H06 7 3.4 M Malonate 0 0 0 0 A07 0.784 M Sodium phosphate 0.016 M Di-potassium 5 monobasic monohydrate hydrogenophosphate A08 0.72 M Sodium phosphate 0.08 M Di-potassium 5.6 monobasic monohydrate hydrogenophosphate A09 0.52 M Sodium phosphate 0.28 M Di-potassium 6.3 monobasic monohydrate hydrogenophosphate A10 0.28 M Sodium phosphate 0.52 M Di-potassium 6.9 monobasic monohydrate hydrogenophosphate A11 0.12 Sodium phosphate 0.68 M Di-potassium 7.5 monobasic monohydrate hydrogenophosphate A12 G.032 M Sodium phosphate 0.768 M Di-potassium 8.2 monobasic monohydrate hydrogenophosphate B07 0.98 M Sodium phosphate 0.02 Di-potassium 5 monobasic monohydrate hydrogenophosphate B08 0.9 Sodium phosphate 0.1 M Di-potassium 5.6 monobasic monohydrate hydrogenophosphate B09 0.65 M Sodium phosphate 0.35 M Di-potassium 6.3 monobasic monohydrate hydrogenophosphate B10 0.35 M Sodium phosphate 0.65 M Di-potassium 6.9 monobasic monohydrate hydrogenophosphate B11 0.15 M Sodium phosphate 0.85 M Di-potassium 7.5 monobasic monohydrate hydrogenophosphate B12 0.04 Sodium phosphate 0.96 M Di-potassium 8.2 monobasic monohydrate hydrogenophosphate C07 1.372 M Sodium phosphate 0.028 M Di-potassium 5 monobasic monohydrate hydrogenophosphate C08 1.26 M Sodium phosphate 0.14 Di-potassium 5.6 monobasic monohydrate hydrogenophosphate C09 0.91 M Sodium phosphate 0.49 M Di-potassium 6.3 monobasic monohydrate hydrogenophosphate C10 0.49 M Sodium phosphate 0.91 M Di-potassium 6.9 monobasic monohydrate hydrogenophosphate C11 0.21 M Sodium phosphate 1.19 M Di-potassium 7.5 monobasic monohydrate hydrogenophosphate C12 0.056 M Sodium phosphate 1.344 M Di-potassium 8.2 monobasic monohydrate hydrogenophosphate 1.764 M Sodium phosphate 0.036 M Di-potassium D07 monobasic monohydrate hydrogenophosphate 5 D08 1.62 M Sodium phosphate 0.18 M Di-potassium 5.6 monobasic monohydrate hydrogenophosphate D09 1.17 M Sodium phosphate 0.63 M Di-potassium 6.3 monobasic monohydrate hydrogenophosphate D10 0.63 M Sodium phosphate 1.17 M Di-potassium 6.9 monobasic monohydrate hydrogenophosphate D11 0.27 Sodium phosphate 1.53 M Di-potassium 7.5 monobasic monohydrate hydrogenophosphate D12 0.072 M Sodium phosphate 1.728 M Di-potassium 8.2 monobasic monohydrate hydrogenophosphate E07 0.1 M CITRIC ACID 4 0.8 M Sodium formate pH4 E08 0.1 M CITRIC ACID 5 0.8 M Sodium formate pH5 E09 0.1 M MES 6 0.8 M Sodium formate pH6 E10 0.1 M HEPES 7 0.8 M Sodium formate pH7 E11 0.1 M TRIS 8 0.8 M Sodium formate pH8 E12 0.1 M BI1NE 9 0.8 M Sodium formate pH9 F07 0 M CITRIC ACID 4 1.6 M Sodium formate pH4 F08 0.1 M CITRIC ACID 5 1.6 M Sodium formate pH5 F09 0.1 M MES 6 1.6 M Sodium formate pH6 F10 0.1 M HEPES 7 1.6 M Sodium formate pH7 F11 0.1 M TRIS 8 1.6 M Sodium formate pH8 F12 0.1 M BICINE 9 1.6 M Sodium formate pH9 G07 0.1 M CITRIC ACID 4 2.4 M Sodium formate pH4 G08 0.1 M CITRIC ACID 5 2.4 M Sodium formate pH5 G09 0.1 M MES 6 2.4 M Sodium formate pH6 G10 0.1 M HEPES 7 2.4 M Sodium formate pH7 G11 0.1 M TRIS 8 2.4 M Sodium formate pH8 G12 0.1 M BICINE 9 2.4 M Sodium formate pH9 H07 0.1 M CITRIC ACID 4 3.2 M Sodium formate pH4 H08 0.1 M CITRIC ACID 5 3.2 M Sodiumformate pH5 H09 0.1 M MES 6 3.2 M Sodium formate pH6 H10 0.1 M HEPES 7 3.2 M Sodium formate pH7 H11 0.1 M TRIS 8 3.2 M Sodium formate pH8 H12 0.1 M BICINE 9 3.2 M Sodium formate pH9 

The invention claimed is:
 1. Lathanide complexes formed of a lanthanide ion Ln³⁺ and a ligand of formula (IA-IC), the lanthanide complexes being cationic and having a positive charge greater than or equal to one, the ligand of formula (IA-IC) forming an open coordination sphere comprising aromatic groups with at least seven coordination sites of the lanthanide ion Ln3+, the ligand of formula (IA-IC) being:

in which: n is equal to 1, 2 or 3; R₂ is a hydrogen atom or a methyl group or —CH₂R₅, where R₅ is a phenyl, or pyridinyl; R₄ represents —H, —CH₃, —CH₂R₆; R₆ represents a phenyl or pyridinyl group; R₁ represents:

 with: R₇ which represents —COO⁻, —CONH₂, —CONHR₉, —PR₉OO⁻, or a selected group chosen from:

with R₉ which represents a hydrogen atom, a methyl, ethyl or phenyl group; and R₈ which represents a hydrogen, fluorine, chlorine, bromine or iodine atom, an —OH or —NH₂; and their salts with an anion, their solvates and hydrates.
 2. Complexes according to claim 1 in the form of a salt with an anion chosen from: Cl⁻, Br⁻, I⁻, OH⁻, NO₃ ⁻, triflate, PF₆ ⁻, SbF₆ ⁻, B(Ph)₄ ⁻, BF₄ ⁻, sulphates, carbonates, phosphates and carboxylates.
 3. Complexes according to claim 1 formed with a ligand corresponding to the formula (IA):

wherein n=1 and R₂=H, and R₁ represents:

as well as their salts with an anion, their solvates and hydrates.
 4. Complexes according to claim 1 characterized in that R₁ represents:

with: R₇ which represents COO⁻ or —PR₉OO⁻ with R₉ which represents a group methyl or ethyl; or R₇ which represents

R₈ which represents a hydrogen, fluorine, chlorine, bromine or iodine atom; as well as their salts with an anion, their solvates and hydrates.
 5. Complexes according to claim 1, chosen from among complexes of formula:

as well as their salts with an anion, in particular their hydrochloride salt, their solvates and hydrates.
 6. Complexes according to claim 1, with a lanthanide ion Ln³⁺, Ln being Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb or Lu, with Eu, Tb, Yb and Lu being preferred. 